Archive for the ‘Crispr’ Category

CRISPR-Cas9 is described as a cut-and-paste technique that targets and removes specific sections of DNA. Different DNA can then be pasted in its place.Flickr/Andy Leppard

Our genes are the basic functional units of heredity that code for proteins which determine our characteristics. Variations in genomes arise due to internal cell processes, such as mistakes made when copying DNA, or external factors such as ultraviolet radiation from sunlight. Beyond this, the ability to selectively and artificially modify genes to add, remove or change traits is a developing field with huge potential but also worrying ethical implications. As of February 2020, new steps have been taken towards using this medical technology in the clinic and so these ethical concerns require urgent attention.

What is CRISPR-Cas9?

CRISPR-Cas9 is described as a cut-and-paste technique that targets and removes specific sections of DNA. Different DNA can then be pasted in its place.

This works using specific regions of DNA called Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR). These DNA sequences are recognised by an endonuclease enzyme called Cas9, using its own guide RNA. This allows Cas9 to cut both strands of DNA at specific points in the genome.

Where did the idea for CRISPR-Cas9 come from?

The principle for CRISPR-Cas9 gene editing was derived from a protection measure in certain bacteria against a group of viruses called bacteriophages. Cas9 retains genetic information from previous virus infections in the form of its guide RNA, which it then compares to foreign viral DNA of subsequent infections. If the foreign DNA matches the RNA, then Cas9 cleaves the invading DNA.

Can CRISPR save lives?

In February 2020, a research team in the USA safely edited the immune systems of three patients suffering from cancer without any negative side effects. CRISPR-Cas9 was used to target multiple genes in T cells, a type of immune cell. The modifications led to certain changes that stimulated the immune system to specifically recognise and attack cancerous cells.

Cancer is the second leading cause of death globally, accounting for over 15% of deaths. Any new approach that could provide a new perspective on cancer treatment is clearly hugely important.

However, there are some disadvantages to consider. Cas9 has a relatively high accuracy rate but the possibility of offshooting still exists - when the target gene is not hit correctly. This can lead to abnormal gene function and expression. Additionally, Cas9 and the RNA it uses to recognise CRISPR sequences is relatively large and so delivery into the nucleus of the cell, where the DNA is located, can be difficult.

What about editing at the germline?

CRISPR-Cas9 can be used to edit genomes at the germline level, meaning that any modifications made will be present in all cells of the organism involved, and will be passed onto offspring. This may initially sound like a good thing - any positive changes made, such as removing a gene variant associated with increased cancer risk, can benefit all future generations with just one change. However, editing at the germline raises complicated ethical questions. Is it morally acceptable to choose what characteristics we give our offspring? Even if this starts in the context of disease prevention, it may well be a slippery slope leading to the removal of any trait viewed as undesirable, pushing society into a eugenics movement - a set of practices that work to improve the genetic quality of the human population.

Many fear that this could widen the gap between the rich and poor, if only some members of society are able to afford CRISPR editing.

He Jiankui and the backlash regarding designer babies

In November 2018, He Jiankui presented results of his work in creating genetically edited babies. Jiankui used CRISPR technology to edit DNA in human embryos to make them less susceptible to HIV. This is an example of how CRISPR can be used to prevent future disease, but Jiankuis actions have been heavily condemned and have led to his prosecution on the grounds that he breached scientific and ethical conduct. The technology employed was deemed too advanced to be used on newborn babies and the risk-benefit ratio was deemed inappropriate, as the babies would not have been immediately at high risk for HIV.

Beyond Healthcare

CRISPR can also be used in agriculture, to create compact plants with less sprawling bushes, larger fruits that can ripen at the same time, higher vitamin C levels and many other characteristics that can either aid in crop growth or improve crop quality. Increased crop quantity could be hugely beneficial as the global population continues to rise and higher vitamin content can help to ensure that the prevalence of deficiency diseases is reduced.

The Future of CRISPR

There is still much scope to improve CRISPR techniques. In Zurich, Switzerland, Cas9 has been replaced with Cas12a (a similar enzyme) that can allow for targeting of lots of genes with a smaller RNA molecule. This is likely to be faster and more efficient, and may help to solve the problem of reaching the DNA in the cells nucleus. Tufts University in the USA has also attempted to improve CRISPR by using a different delivery method for Cas9 that could allow it to diffuse straight through the cell membrane.

Even if we were to achieve optimal function of the CRISPR-Cas 9 technique, this does not mean that CRISPR use is undisputedly positive. Gene editing techniques come with a range of ethical challenges, many of which may remain unresolved even as the technology is coming ever closer to widespread use.

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CRISPR-Cas9: Should we be able to edit our genes? - Varsity Online

Legendary investor Warren Buffett advises to be fearful when others are greedy, and be greedy when others are fearful. One way we can try to measure the level of fear in a given stock is through a technical analysis indicator called the Relative Strength Index, or RSI, which measures momentum on a scale of zero to 100. A stock is considered to be oversold if the RSI reading falls below 30.

In trading on Thursday, shares of CRISPR Therapeutics AG (Symbol: CRSP) entered into oversold territory, hitting an RSI reading of 29.6, after changing hands as low as $38 per share. By comparison, the current RSI reading of the S&P 500 ETF (SPY) is 27.7. A bullish investor could look at CRSP's 29.6 RSI reading today as a sign that the recent heavy selling is in the process of exhausting itself, and begin to look for entry point opportunities on the buy side. The chart below shows the one year performance of CRSP shares:

Looking at the chart above, CRSP's low point in its 52 week range is $33.55 per share, with $74 as the 52 week high point that compares with a last trade of $40.11.

Find out what 9 other oversold stocks you need to know about

The views and opinions expressed herein are the views and opinions of the author and do not necessarily reflect those of Nasdaq, Inc.

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CRISPR Therapeutics Enters Oversold Territory (CRSP) - Nasdaq

Shares of Crispr Therapeutics AG (NASDAQ:CRSP) have been assigned a consensus rating of Buy from the twenty ratings firms that are presently covering the stock, Marketbeat Ratings reports. Two research analysts have rated the stock with a sell recommendation, four have assigned a hold recommendation and twelve have assigned a buy recommendation to the company. The average 12 month target price among brokers that have issued a report on the stock in the last year is $74.42.

Several equities analysts have commented on CRSP shares. Stifel Nicolaus started coverage on Crispr Therapeutics in a research report on Wednesday, March 4th. They set a hold rating and a $52.00 price objective for the company. Citigroup raised their price objective on Crispr Therapeutics from $28.00 to $31.00 and gave the company a sell rating in a research report on Wednesday, March 4th. Canaccord Genuity raised their price objective on Crispr Therapeutics from $72.00 to $80.00 and gave the company a positive rating in a research report on Wednesday, November 20th. Needham & Company LLC reissued a buy rating and set a $84.00 price objective on shares of Crispr Therapeutics in a research report on Monday, December 23rd. Finally, Piper Jaffray Companies reissued a buy rating and set a $104.00 price objective on shares of Crispr Therapeutics in a research report on Monday, December 16th.

Large investors have recently modified their holdings of the company. Farmers & Merchants Trust Co of Chambersburg PA bought a new position in shares of Crispr Therapeutics during the fourth quarter valued at $26,000. Webster Bank N. A. bought a new position in shares of Crispr Therapeutics during the fourth quarter valued at $26,000. Advisory Services Network LLC increased its holdings in shares of Crispr Therapeutics by 146.0% during the fourth quarter. Advisory Services Network LLC now owns 674 shares of the companys stock valued at $41,000 after acquiring an additional 400 shares in the last quarter. Clear Harbor Asset Management LLC bought a new position in shares of Crispr Therapeutics during the fourth quarter valued at $44,000. Finally, Exchange Traded Concepts LLC bought a new position in shares of Crispr Therapeutics during the fourth quarter valued at $54,000. Hedge funds and other institutional investors own 52.04% of the companys stock.

Crispr Therapeutics (NASDAQ:CRSP) last announced its quarterly earnings data on Wednesday, February 12th. The company reported $0.51 EPS for the quarter, beating the consensus estimate of ($0.68) by $1.19. The business had revenue of $77.00 million during the quarter, compared to analyst estimates of $39.08 million. Crispr Therapeutics had a return on equity of 11.74% and a net margin of 23.09%. The companys revenue for the quarter was up 76900.0% on a year-over-year basis. During the same quarter last year, the firm posted ($0.92) earnings per share. As a group, equities analysts expect that Crispr Therapeutics will post -4.54 EPS for the current year.

Crispr Therapeutics Company Profile

CRISPR Therapeutics AG, a gene editing company, focuses on developing transformative gene-based medicines for the treatment of serious human diseases using its regularly interspaced short palindromic repeats associated protein-9 (CRISPR/Cas9) gene-editing platform in Switzerland. Its lead product candidate is CTX001, an ex vivo CRISPR gene-edited therapy for treating patients suffering from dependent beta thalassemia or severe sickle cell disease in which a patient's hematopoietic stem cells are engineered to produce high levels of fetal hemoglobin in red blood cells.

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Crispr Therapeutics AG (NASDAQ:CRSP) Receives Consensus Recommendation of Buy from Brokerages - Redmond Register

NORGES BANK INVESTMENT MANAGEMEN bought a fresh place in Boston Properties Inc. (NYSE:BXP). The institutional investor bought 2.4 million shares of the stock in a transaction took place on 12/31/2019. In another most recent transaction, which held on 12/31/2019, SECURITY CAPITAL RESEARCH & MANA bought approximately 832.5 thousand shares of Boston Properties Inc. In a separate transaction which took place on 12/31/2019, the institutional investor, WELLINGTON MANAGEMENT CO. LLP bought 425.7 thousand shares of the companys stock. The total Institutional investors and hedge funds own 99.00% of the companys stock.

In the most recent purchasing and selling session, Boston Properties Inc. (BXP)s share price decreased by -7.75 percent to ratify at $111.89. A sum of 1868448 shares traded at recent session and its average exchanging volume remained at 760.61K shares. The 52-week price high and low points are important variables to concentrate on when assessing the current and prospective worth of a stock. Boston Properties Inc. (BXP) shares are taking a pay cut of -24.31% from the high point of 52 weeks and flying high of -6.89% from the low figure of 52 weeks.

Boston Properties Inc. (BXP) shares reached a high of $117.45 and dropped to a low of $108.11 until finishing in the latest session at $112.69. Traders and investors may also choose to study the ATR or Average True Range when concentrating on technical inventory assessment. Currently at 5.60 is the 14-day ATR for Boston Properties Inc. (BXP). The highest level of 52-weeks price has $147.83 and $120.17 for 52 weeks lowest level. After the recent changes in the price, the firm captured the enterprise value of $32.94B, with the price to earnings ratio of 33.92 and price to earnings growth ratio of 4.85. The liquidity ratios which the firm has won as a debt-to-equity ratio of 2.19.

Having a look at past record, were going to look at various forwards or backwards shifting developments regarding BXP. The firms shares fell -18.17 percent in the past five business days and shrunk -22.42 percent in the past thirty business days. In the previous quarter, the stock fell -19.86 percent at some point. The output of the stock decreased -15.25 percent within the six-month closing period, while general annual output lost -17.14 percent. The companys performance is now negative at -18.84% from the beginning of the calendar year.

According to WSJ, Boston Properties Inc. (BXP) obtained an estimated Overweight proposal from the 21 brokerage firms currently keeping a deep eye on the stock performance as compares to its rivals. 0 equity research analysts rated the shares with a selling strategy, 8 gave a hold approach, 12 gave a purchase tip, 1 gave the firm a overweight advice and 0 put the stock under the underweight category. The average price goal of one year between several banks and credit unions that last year discussed the stock is $151.00.

CRISPR Therapeutics AG (CRSP) shares on Thursdays trading session, dropped -11.92 percent to see the stock exchange hands at $38.12 per unit. Lets a quick look at companys past reported and future predictions of growth using the EPS Growth. EPS growth is a percentage change in standardized earnings per share over the trailing-twelve-month period to the current year-end. The company posted a value of $0.97 as earning-per-share over the last full year, while a chance, will post -$4.99 for the coming year. The current EPS Growth rate for the company during the year is 134.10% and predicted to reach at -10.40% for the coming year. In-depth, if we analyze for the long-term EPS Growth, the out-come was 54.00% for the past five years.

The last trading period has seen CRISPR Therapeutics AG (CRSP) move -48.49% and 13.62% from the stocks 52-week high and 52-week low prices respectively. The daily trading volume for CRISPR Therapeutics AG (NASDAQ:CRSP) over the last session is 1.48 million shares. CRSP has attracted considerable attention from traders and investors, a scenario that has seen its volume jump 37.35% compared to the previous one.

Investors focus on the profitability proportions of the company that how the company performs at profitability side. Return on equity ratio or ROE is a significant indicator for prospective investors as they would like to see just how effectively a business is using their cash to produce net earnings. As a return on equity, CRISPR Therapeutics AG (NASDAQ:CRSP) produces 11.70%. Because it would be easy and highly flexible, ROI measurement is among the most popular investment ratios. Executives could use it to evaluate the levels of performance on acquisitions of capital equipment whereas investors can determine that how the stock investment is better. The ROI entry for CRSPs scenario is at 4.90%. Another main metric of a profitability ratio is the return on assets ratio or ROA that analyses how effectively a business can handle its assets to generate earnings over a duration of time. CRISPR Therapeutics AG (CRSP) generated 9.60% ROA for the trading twelve-month.

Volatility is just a proportion of the anticipated day by day value extendthe range where an informal investor works. Greater instability implies more noteworthy benefit or misfortune. After an ongoing check, CRISPR Therapeutics AG (CRSP) stock is found to be 8.52% volatile for the week, while 7.29% volatility is recorded for the month. The outstanding shares have been calculated 63.90M. Based on a recent bid, its distance from 20 days simple moving average is -27.05%, and its distance from 50 days simple moving average is -31.04% while it has a distance of -25.29% from the 200 days simple moving average.

The Williams Percent Range or Williams %R is a well-known specialized pointer made by Larry Williams to help recognize overbought and oversold circumstances. CRISPR Therapeutics AG (NASDAQ:CRSP)s Williams Percent Range or Williams %R at the time of writing to be seated at 99.28% for 9-Day. It is also calculated for different time spans. Currently for this organization, Williams %R is stood at 99.28% for 14-Day, 99.48% for 20-Day, 99.54% for 50-Day and to be seated 99.67% for 100-Day. Relative Strength Index, or RSI(14), which is a technical analysis gauge, also used to measure momentum on a scale of zero to 100 for overbought and oversold. In the case of CRISPR Therapeutics AG, the RSI reading has hit 24.91 for 14-Day.

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Stocks Expected To Recuperate: Boston Properties Inc. (BXP) and CRISPR Therapeutics AG (CRSP) - BOV News

Warren Buffett once said: "Opportunities come infrequently. When it rains gold, put out the bucket, not the thimble." Many investors don't realize it, but it's raining gold right now.

The stock market sell-off caused by worries about the coronavirus outbreak (and, more recently, plunging oil prices) has made a lot of really good stocks cheaper than they've been in quite a while. Long-term investors with available cash should be getting their buckets ready to put out. Here are three stocks where you could invest $10,000 right now and likely reap significant returns over the long run.

Image source: Getty Images.

Vertex Pharmaceuticals (NASDAQ:VRTX) haven't fallen as much as most other stocks during the coronavirus correction. That makes sense. Biotech stocks shouldn't be negatively affected by COVID-19, generally speaking. The companies' drugs will still be needed. Their clinical studies of drug candidates will still move forward.

You can add an exclamation point to those statements when it comes to Vertex. The biotech dominates the worldwide cystic fibrosis (CF) market. Over the last few months, it's picked up important reimbursement deals in Europe and elsewhere that should boost sales for several of its CF drugs. Vertex already won U.S. approval for Trikafta, its newest CF drug on the market, and awaits approval in Europe. The drug should expand the addressable patient population for its CF therapies by more than 50%.

Vertex's pipeline is focused on several other rare genetic diseases. It's partnering with CRISPR Therapeuticsto test gene-editing therapies for treating beta-thalassemia and sickle cell disease. It's evaluating VX-814 in a phase 2 clinical study targeting alpha-1 antitrypsin deficiency. Vertex also has preclinical and early stage clinical programs targeting other rare genetic diseases.

But the biotech doesn't have its eyes only on rare diseases. Vertex is developing drugs that manage pain. Thanks to its acquisition of Semma Therapeutics last year, it's also now on a path to advance an experimental drug to clinical testing that could potentially cure type 1 diabetes.

The traditional way of buying advertising is for agencies to negotiate over a period of time with media companies.The Trade Desk (NASDAQ:TTD) operates a software platform that allows advertisers to buy digital ads immediately and cost-effectively through what's called programmatic ad buying.

Shares of The Trade Desk have dropped more than 25% from the highs set earlier this year. Have the business prospects for the company dwindled because of the coronavirus epidemic? No.

It's possible that some companies could be impacted by COVID-19 and cut back on their advertising budgets. However, it's also likely that more people will stay home, watch streaming TV, and browse the internet more instead of getting out and exposing themselves to infection by the novel coronavirus.

The Trade Desk beat Wall Street estimates in its Q4 results, delivering year-over-year revenue growth of 35% and adjusted earnings growth of 37%. The company also provided an optimistic outlook for 2020 despite its management being fully aware of the COVID-19 threat. With the programmatic ad market still only in its early stages and The Trade Desk standing atop the industry, the current market correction presents a tremendous buying opportunity.

Enterprise Products Partners (NYSE:EPD) stock has plunged nearly 40% since early January. The entire oil and gas industry has been hit by a double-whammy. The coronavirus outbreak is causing people to travel less, reducing demand for oil. More importantly, an oil price war triggered by Russia is hammering North American oil and gas companies especially hard.

I don't expect that Enterprise Products Partners stock will recover until oil prices rebound somewhat. But I think that's going to happen sooner or later. Russia (and Saudi Arabia) won't be able to flood the market with cheap oil indefinitely.

Keep in mind that Enterprise Products Partners' natural gas pipelines, storage, transportation, and processing businesses make most of their money from fees and not commodity-based pricing. That will help the company move past the current challenges. It's also beneficial that Enterprise Products Partners ranks as one of the bellwethers in the midstream market and has a solid credit profile.

Buying shares of Enterprise Products Partners right now might seem a little scary, but there's a nice bonus -- the company's dividend. Thanks to the shellacking its stock has taken, Enterprise Products Partners' dividend yield stands at close to 9.8%.

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Coronavirus Market Correction: Where to Invest $10000 Right Now - Motley Fool

Researchers from the University of Maryland have created a new CRISPR gene-editing system that can successfully modify the DNA of plants to a greater extent than was ever possible before.

The new system, referred to as CRISPR-Cas12b, would allow scientists to effectively modify crops for various purposes, such as making them more resistant to diseases or pests. Previous gene-editing systems, such as CRISPR-Cas9 and CRISPR-Cas12a, have received plenty of attention in the scientific community, but aren't as well-suited to modifying plant DNA.

Image source: Getty Images.

"This type of technology helps increase crop yield and sustainably feed a growing population in a changing world. In the end, we are talking about broad impact andpublic outreach, because we need to bridge the gap between what researchers are doing and how those impacts affect the world," said Yiping Qi, assistant professor of plant science at the University of Maryland and the creator of the CRISPR-Cas12b system.

Ever since CRISPR technology first came onto the scene, researchers have considered using it to genetically modify crops. Scientists are already experimenting with CRISPR technology to make bananas that are more resistant to a strain of deadly fungus that is ravaging plantations in Latin America.

However, using CRISPR to treat conditions in humans remains its most compelling application. Many gene-editing biotech companies, among them CRISPR Therapeutics (NASDAQ:CRSP) and Editas Medicine, are alreadymaking use of CRISPR-Cas9 technology as they develop treatments for use in humans.

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Researchers Create New CRISPR Gene-Editing System for Plants - The Motley Fool

The rapid and frightening spread of the coronavirus has sparked a battle thats drawing on a host of emerging technologies. Government, industry and academic researchers are scrambling to improve our ability to diagnose, treat and contain a virus thats threatening to reach pandemic status.

This isnt the first time researchers have faced off against a dangerous member of this family of viruses. But it is the first time theyve done it with a toolbox that includes the gene-editing tool CRISPR and the emerging field of synthetic biology.

Indeed, weve known about coronaviruses for nearly 60 years. But for several decades, they attracted little attention, causing symptoms similar to the common cold.

That changed in 2003, when a deadly member of the coronavirus family, SARS-COV, spread to 29 countries, killing 774 people. Suddenly, a coronavirus found previously in animals had managed to jump to humans, where it killed nearly 10 percent of those infected. The virus sparked fear across the globe, but was brought under control within a year. Only a small number of cases have been reported since 2004.

Then in 2012 came MERS-COV. The virus emerged in Saudi Arabia, jumping from camels to humans. The virus has never caused a sustained outbreak, but with a mortality rate of35 percent, it has killed 858 people so far. Infections have been reported in 27 countries, with most in the Middle East. The virus is considered by the World Health Organization to be a potential epidemic threat.

Interestingly, neither of these previous coronavirus threats were stopped by a cure or a vaccine. MERS still lurks in the background, while SARS was contained by what amounts to old-school practices, according to a 2007 article in Harvard Magazine:

Ironically, in this age of high-tech medicine, the virus was eventually brought under control by public-health measures typically associated with the nineteenth centuryisolation of SARS patients themselves and quarantine of all their known and suspected contactsrather than a vaccine.

There currently is no cure for this new wave of coronavirus infections (the resulting disease is called Covid-19), even though some antiviral therapies are being tested and one experimental vaccine is ready for testing in humans. The virus genome has been sequenced and its genetic code may shed light on how the disease starts and spreads, as well as inform on potential pharmaceutical targets for drug development. The Covid-19 virus similarity to the SARS-COV may mean that cures developed for one strain may prove effective for the other. The Canadian company AbCellera plans to test its antibody technology, already tried against MERS-COV, to neutralize the Covid-19 viral bodies.

What is really encouraging is the level of international collaboration aimed to fight this health emergency. Funding bodies, scientific societies and scientific journals have signed a joint statement, agreeing to openly share research findings with the global research community as soon as they are available. The very quick information dissemination gave scientists around the globe several RNA sequences of the virus genome. And these sequences can be used to better understand the epidemiology and origins of the virus. Moreover, the advancements in DNA technology let research groups in academia and industry synthesize the viral genetic material to use in the two areas of focus: detection of virus and vaccine development.

One of the trickiest things about the coronavirus is its speculated transmission by asymptomatic patients. This increases the number of infections and makes containment measures less effective, spreading fears that the virus may establish a permanent presence in some areas. There are also fears that many incidents lie undetected, spreading the virus under the radar. As of March 9, the virus has infected more than 110,000 people, killing nearly 4,000, in 97 countries.

Several biotech companies have scrambled to provide kits and resources for early and reliable detection of the new coronavirus. Mammoth Bioscience, a San Francisco-based startup, is already working on a detection assay using their CRISPR technology. The DNA technology companies IDT and Genscript already distribute PCR-based kits for detection for research purposes. The Chinese companies BGI and Liferiver Biotech use the same PCR technology for the kits they provide to their countries health authorities.

The French-British biotech Novacyt announced the launch of a diagnostic kit for clinical use in middle February. The kit will also use quantitative-PCR, developed by their sister company Primerdesign. Its high specificity will reduce the analysis time to less than two hours. The companys CEO Graham Mullis told Reuters that each kit will cost around $6.50, and that they have already received more than 33,000 orders.

The only way to effectively control and even eliminate the outbreak is to develop a vaccine. Unfortunately, the new outbreak hasnt attracted the attention of the lead vaccine manufacturers. Non-profit organizations, such as the Coalition for Epidemic Preparedness Innovations (CEPI), have jumped in to fill the gap. But despite the emergency, a vaccine may be several years away from being available

The University of Queensland in Brisbane, Australia, announced that theyre working on a coronavirus vaccine which they hope to have ready within the next few months. The molecular clamp approach the Australian researchers have developed allows is designed to boost the immune system response and work against several viral infections. GlaxoSmithKline has offered is adjuvant technology adjuvants are added to vaccines to boost their efficiency to speed up the process.

The Cambridge, MA-based Moderna uses a different approach to make vaccines. Their mRNA technology is modular and very adaptable to use for a new disease or when the epitope (the vaccines target) mutates. The company says its vaccine is ready for human trials.

The Covid-19 outbreak has rightly gained the attention of health authorities and the media. If the virus were to reach countries with weaker healthcare systems than Chinas, the number of deaths will rise significantly and containment will be even harder. Moreover, the long incubation time of the disease, combined with the asymptomatic spread, make quarantine and isolation measures less effective. The biggest risk is for the new coronavirus to become endemic in certain areas, where the disease is never truly extinct and displays seasonal outbreaks. We dont want the Covid-19 to become a new flu.

The health authorities of 2020, the biotech industry, and the society in general are better prepared for a coronavirus outbreak than a few years ago. The situation is less risky than MERS and SARS, though the new virus is harder to contain. This outbreak offers a chance for everyone to become more aware of viral infections, the appropriate precautions and get vaccinated according to the official recommendations. And keep in mind that the best way to stay informed is through official sources, such as the WHO and the CDC.

As for the biotech industry, are they playing their part? The answer is a partial yes; there are several companies that immediately scrambled to help the situation. But the big players within the field could be doing more.

Kostas Vavitsas, PhD, is a Senior Research Associate at the University of Athens, Greece. He is also a steering committee member of EUSynBioS. Follow him on Twitter @konvavitsas

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Fighting the coronavirus outbreak with genetic sequencing, CRISPR and synthetic biology - Genetic Literacy Project

The recent performance of CRISPR Therapeutics AG (NASDAQ:CRSP) stock in the market spoke loud and clear to investors as CRSP saw more than 1.08M shares in trading volumes in the last trading session, way higher than the average trading volume of 1.08M shares by far recorded in the movement of CRISPR Therapeutics AG (CRSP). At the time the stock opened at the value of $52.64, making it a high for the given period, the value of the stock dropped by -4.77%. After the decrease, CRSP touched a low price of $49.1553, calling it a day with a closing price of $53.41, which means that the price of CRSP went 50.86 below the opening price on the mentioned day.

Given the most recent momentum in the market in the price movement of CRSP stock, some strong opinions on the matter of investing in the companys stock started to take shape, which is how analysts are predicting an estimated price of $74.46 for CRSP within consensus. The estimated price would demand a set of gains in total of -0.34%, which goes higher than the most recent closing price, indicating that the stock is in for bullish trends. Other indicators are hinting that the stock could reach an outstanding figure in the market share, which is currently set at 48.07M in the public float and 3.25B US dollars in market capitalization.

When it comes to the technical analysis of CRSP stock, there are more than several important indicators on the companys success in the market, one of those being the Relative Strength Indicator (RSI), which can show, just as Stochastic measures, what is going on with the value of the stock beneath the data. This value may also indicate that the stock will go sideways rather than up or down, also indicating that the price could stay where it is for quite some time. When it comes to Stochastic reading, CRSP stock are showing 42.28% in results, indicating that the stock is neither overbought or oversold at the moment, providing it with a neutral within Stochastic reading as well. Additionally, CRSP with the present state of 200 MA appear to be indicating bearish trends within the movement of the stock in the market. While other metrics within the technical analysis are due to provide an outline into the value of CRSP, the general sentiment in the market is inclined toward negative trends.

With the previous 100-day trading volume average of 780882 shares, Paycom Software (PAYC) recorded a trading volume of 758060 shares, as the stock started the trading session at the value of $282.26, in the end touching the price of $267.71 after dropping by -5.15%.

Paycom Software (PAYC) surprised the market during the previous quarter closure with the last reports recording $0.78, compared to the consensus estimation that went to $0.70. The records showing the total in revenues marked the cap of +28.65%, which means that the revenues increased by +44.51% since the previous quarterly report.

PAYC stock seem to be going ahead the lowest price in the last 52 weeks with the latest change of 58.35%.Then price of PAYC also went backward in oppose to its average movements recorded in the previous 20 days. The price volatility of PAYC stock during the period of the last months recorded 4.63%, whilst it changed for the week, now showing 6.09% of volatility in the last seven days. The trading distance for this period is set at -9.79% and is presently away from its moving average by -9.32% in the last 50 days. During the period of the last 5 days, PAYC stock lost around -5.29% of its value, now recording a sink by 8.54% reaching an average $246.92 in the period of the last 200 days.During the period of the last 12 months, Paycom Software (PAYC) jumped by 1.11%.

According to the Barcharts scale, the companys consensus rating was unchanged to 3.92 from 3.92, showing an overall improvement during the course of a single month.

PAYC shares recorded a trading volume of 1.03 million shares, compared to the volume of 881.54K shares before the last close, presented as its trading average. With the approaching 6.09% during the last seven days, the volatility of PAYC stock remained at 4.63%. During the last trading session, the lost value that PAYC stock recorded was set at the price of $267.71, while the lowest value in the last 52 weeks was set at $169.06. The recovery of the stock in the market has notably added 58.35% of gains since its low value, also recording -16.99% in the period of the last 1 month.

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This Is (Another) Opportunity To Buy CRISPR Therapeutics AG (CRSP), Paycom Software (PAYC) - US Post News

INTRODUCTION

An efficient gene drive could rapidly modify or suppress a target population (14). Such a mechanism could potentially be used to prevent transmission of vector-borne diseases such as malaria or dengue and could also have conservation applications (14). The best-studied form of an engineered gene drive mechanism is the homing drive, which uses the CRISPR-Cas9 system to cleave a wild-type allele. The drive allele is then copied into the wild-type site via homology-directed repair, increasing the frequency of the drive allele in the population. Thus far, CRISPR homing gene drives have been demonstrated in yeast (58), flies (916), mosquitoes (1719), and mice (20).

However, homing drives typically suffer from high rates of resistance allele formation. These alleles can form when DNA is repaired by end joining, which often mutates the sequence. The consequence of a changed sequence is that the drives guide RNA (gRNA) can no longer target the allele for cleavage. Resistance alleles have been observed to arise both in germline cells as an alternative to homology-directed repair and in the early embryo due to deposition of Cas9 and gRNA into the egg by drive-carrying mothers (12). While formation of resistance alleles remains the primary obstacle to construction of efficient gene drives, substantial progress has been made toward overcoming this challenge. For example, a suppression-type drive in Anopheles gambiae (21) and a modification-type drive in Drosophila melanogaster (16) avoided issues with resistance alleles by targeting an essential gene. Because of this, resistance alleles that disrupted the function of the target gene had substantially reduced fitness. This allowed both drives to successfully spread through cage populations.

Multiplexing gRNAs has been proposed as a mechanism for increasing the efficiency of gene drives (1, 4). This would purportedly work by two mechanisms. First, having multiple cut sites would potentially allow drive conversion even if some of the sites have resistance sequences due to previous end joining repair at those sites. As long as at least one site remains wild type and, thus, cleavable, homology-directed repair can still occur. Second, the chance of forming a full resistance allele that preserves the function of the target gene would be substantially reduced due to the possibility of disruptive mutations forming at any of the gRNA target sites. Resistance alleles that disrupt the function of the target gene incur large fitness costs in several drive designs, which would make resistance substantially less likely to block the spread of the drive.

However, two studies using two gRNAs (13, 16) showed somewhat lower increases in efficiency than predicted by simple models of multiple gRNAs (2224). This is partially because most models assume that cleavage and repair by either homology-directed repair or end joining occurs sequentially at each gRNA target site. However, it appears that some germline resistance alleles form before the narrow temporal window for homology-directed repair (10, 12, 13). Additional resistance sequences may also form as a direct alternative to successful homology-directed repair, while others could form after meiosis I when only end joining repair is possible. Furthermore, unless cleavage occurs in both of the outermost gRNA target sites during the window for homology-directed repair, the wild-type chromosome on either side of the cleavage would have imperfect homology to the drive allele because of nonhomologous DNA between the cut site and the homology arm (13). Imperfect homology likely reduces repair fidelity (i.e., less homology-directed repair and more end joining repair). This proposition is supported by the greatly reduced efficiency seen in a construct with four gRNA targets located far apart from one another (9). Last, it is unlikely that gRNAs are the limiting factor in Cas9/gRNA enzymatic activity (15). As the number of gRNAs increases, the total cleavage rate likely plateaus, thus reducing the cleavage rate at each individual site and thereby preventing further gains in drive efficiency.

Here, we systematically model these factors and show how they are expected to affect the performance of homing drives with multiple gRNAs. We verify and parameterize these models via experimental analysis of several homing drives in D. melanogaster. We additionally consider other factors that could reduce gene drive performance, such as partial homology-directed repair and uneven activity of gRNAs. We then apply our model to predict drive performance in Anopheles mosquitoes, assessing several types of homing drives for population modification or suppression. We find that the reduction in efficiency due to imperfect homology is synergistic, with the lower per-site cleavage rates from Cas9 activity saturation. Because of this, each type of drive has an optimal number of gRNAs that results in maximized overall performance, a finding that could inform future designs of homing gene drives.

In our model, we consider five types of homing gene drive systems:

1) Standard drive. The standard homing drive is a population modification system. Its primary drive mechanism occurs in germline cells during early meiosis. When it operates successfully, the drive allele replaces wild-type alleles in the germline. However, resistance alleles can also form, preventing the spread of the drive.

2) Population suppression drive. This drive increases in frequency in the same manner as the standard homing drive, and resistance alleles develop under the same circumstances. However, the drive targets a recessive female fertility gene and disrupts the function of the gene with its presence. Resistance alleles can also disrupt the function of the target gene. Females with two disrupted copies of the gene are rendered sterile, while males are unaffected. Notably, unlike the standard homing drive, this drive does not carry any payload, as, it accomplishes its goal simply with its presence. Such a drive was successful in laboratory populations of the mosquito A. gambiae (21).

3) Haplolethal drive. This drive system is a modification of the standard homing drive system. It targets a gene that is critical to the viability of the individual. However, the drive contains a recoded portion of the gene that is immune to Cas9 cleavage, so the presence of the drive does not disrupt the function of the target. If an individual receives a resistance allele that disrupts the haplolethal target, then that individual will not be viable, preventing these resistance alleles from entering the population. A haplolethal homing drive was successful in a laboratory population of the fruit fly D. melanogaster (16).

4) Recessive lethal drive. This drive is similar to the haplolethal drive, but the target is recessive lethal. Only individuals carrying two resistance alleles that disrupt the target gene function are nonviable. Thus, resistance alleles are removed from the population more slowly. However, this drive may be easier to engineer because the drive can provide rescue even in the presence of a resistance allele. It is also more tolerant of a high rate of embryo resistance allele formation because this allows it to operate better as a toxin-antidote system (25, 26).

5) Gene disruption drive. The gene disruption homing drive is a population modification system that is similar to the suppression drive in that its presence disrupts the target gene, as do resistance alleles. However, individuals with two disrupted copies of this gene remain viable and fertile, although they suffer from a small additional fitness cost. The purpose of this drive is to remove the functionality of a particular gene from the population, which can provide benefits such as reduction in disease transmission (27, 28). An advantage of this drive is that there is no need for a recoded sequence. However, finding suitable targets for particular applications could potentially be difficult.

We implemented each of the gene drive models using SLiM version 3.2.1 (29). SLiM is an individual-based, forward-time population genetic simulation framework. General parameters and ecology components are shared across all models.

Our model considers a single panmictic population of sexually reproducing diploid individuals with nonoverlapping generations. The model differs from a standard Wright-Fishertype model in that population size is not regulated. Offspring are generated from random pairings throughout the population, with mate choice and female fecundity affected by genotype fitness. To determine mate choice, first, a random male is selected. This candidate is then accepted at a rate equal to his genotype fitness (e.g., a male with a fitness of 0.5 is accepted half of the time). If he is rejected, then another random candidate is selected, until either a mate has been chosen or the female fails to find an acceptable mate after a total of 10 attempts. Female fecundity is then multiplied by her genotype fitness, along with a factor representing the impact of population density in the system: 10/(1 + 9 N/K), where N is the total population and K is the carrying capacity. A number of offspring are then generated on the basis of a binomial distribution with a maximum of 50 and p = fecundity/25. This model produces logistic dynamics while allowing the population size to fluctuate around the expected capacity. After pairings and offspring have been determined, the genotypes of the offspring are modified according to the genetic component of the model.

In one set of simulations, a small number of drive/wild-type heterozygous flies were introduced into a wild-type population of 100,000 at an initial frequency of 1%. The simulation was then conducted for 100 generations. In another set of simulations, a wild-type female was crossed to a drive/wild-type heterozygote male, and a configurable number of offspring were generated from that single pairing. The genotype of each offspring was recorded to estimate drive performance parameters. Drive conversion was equal to the fraction of wild-type alleles in the germline converted to drive alleles, and resistance allele formation rates also represented rates of conversion from wild-type alleles. The genetic module of our model is described in the Supplementary Methods.

Detailed descriptions of our plasmid construction techniques, the construction and sequencing primers used, the generation of transgenic lines, fly rearing, phenotyping and analysis techniques, and genotyping are available in the Supplementary Methods.

To compare our results to previous work, we constructed a simple model of homing drive dynamics. This model considers each gRNA site independently, with parameters inspired by highly efficient homing drives in Anopheles mosquitoes (18, 19, 21, 30). At each gRNA target site, we assume a cut rate of 99%. If the site is cut, then there is a 7.8% chance that a resistance sequence will be formed. Otherwise, homology-directed repair occurs, and the entire allele (including all target sites, even if some have resistance sequences) is converted to a drive allele. In this model, increasing the number of gRNA target sites always increases the efficiency of the drive, and arbitrarily low resistance rates can be achieved by adding more target sites (Fig. 1). Since even relatively few gRNAs can reduce resistance allele formation to very low levels under this model, gRNA multiplexing has been considered as a highly promising and comparatively straightforward method to avert resistance in homing gene drives (2224).

Five million offspring were generated from crosses between drive/wild-type heterozygotes and wild-type individuals for each model and number of gRNAs. The rate at which wild-type alleles are converted to resistance alleles in the germline of drive/wild-type heterozygous individuals is shown.

The simple model does not take timing of cleavage and repair into account. However, several lines of evidence indicate that cleavage timing can play a key role in drive conversion. Experiments indicated that wild-type alleles can only be converted to resistance alleles and not to drive alleles in the early embryo due to maternally deposited Cas9 (12, 13), where homology-directed repair for the purposes of drive conversion does not take place at appreciable rates. Furthermore, at least some resistance alleles form in pregonial germline cells that can affect the genotype of multiple offspring (10, 12, 13). After the chromosomes separate in late meiosis, homology-directed repair is no longer possible, and any cleavage at this time results in the formation of resistance alleles by end joining repair. It is thus likely that there is only a narrow temporal window in the germline, during which the drive can be successfully copied via homology-directed repair. This window presumably covers early meiosis when homologous chromosomes are close together, which would increase the chance that one chromosome could be used as a template for repair of a double-strand break in the other.

To explore the expected impact of these mechanisms on resistance rates, we constructed a model where cuts during a homology-directed repair phase occur simultaneously, and the DNA then has a single opportunity to undergo homology-directed repair. In this model, there are discrete temporal phases. In each phase, wild-type gRNA sites are cut before any repair takes place. In the first phase, end joining repair always occurs after cutting, so having more gRNAs allows more target sites to avoid being converted to resistance alleles. Only in the next phase is homology-directed repair possible, which takes place at a specific rate if any cutting occurs. If homology-directed repair (successful drive conversion) does not take place, then end joining is assumed to repair the cut, forming a resistance allele. Thus, as the probability of the DNA being cut approaches 100% due to many gRNAs, the overall rate of resistance formation does not decrease indefinitely. Instead, it reaches a minimum value equal to the chance that end joining takes place instead of successful homology-directed repair in the second phase (Fig. 1). This suggests that the simple model of multiple gRNAs is likely inadequate to accurately assess homing drive dynamics.

Previous experiments with two gRNAs resulted in a lower-efficiency improvement than even that predicted by our model that included timing (13). This was shown even more starkly with a four-gRNA drive (9) that had a lower drive conversion efficiency than a one-gRNA drive. We hypothesize that two additional factors could account for this discrepancy. First, the rate at which homology-directed repair occurs after cleavage in the appropriate phase (which we refer to as repair fidelity) is likely reduced if the DNA on either side of the cut sites does not have immediate homology to the drive (meaning that end resection must proceed for several nucleotides before it reaches DNA with homology to the drive). This will often be the case because a drive allele is constructed to have DNA homologous only to that at the outermost cut sites (the leftmost and rightmost sites). Thus, drive efficiency is reduced except when both outer gRNAs are cleaved. Second, the amount of Cas9 enzyme is limited. Thus, as the number of gRNAs increases, Cas9 eventually becomes saturated with gRNAs and cleavage activity plateaus. This has the effect of decreasing the cleavage rate at individual gRNA sites as the total number of gRNAs increases. To test the impact of these two mechanisms on drive efficiency, we conducted a series of experiments.

We first constructed a drive system in D. melanogaster that targeted a synthetic enhanced green fluorescent protein (EGFP) site with one gRNA and Cas9 driven by the nanos promoter (fig. S1), similar to previous synthetic target site drives (15). Drive/wild-type heterozygotes displayed a drive conversion efficiency of 83% [95% confidence interval (CI), 79 to 86%] in females and 61% (95% CI, 57 to 65%) in males (data S1). These values were higher than previous synthetic target site drives (15), likely due to the different genomic location of the target site or the different gRNA target, which targeted further away from the 3xP3 promoter in EGFP. Drive conversion efficiency was significantly higher in females (P < 0.0001, Fishers exact test), which was consistent with previous studies (13, 15). It remains unclear why this may be the case, but it may be due to sex differences in levels of repair proteins in the germline (possibly related to the lack of male chromosomal recombination), resulting in a higher ratio of homology-directed repair to end joining in the appropriate temporal window. Expression of Cas9 by the nanos promoter could also be variable between the sexes. If expression started earlier in males, then an increased number of resistance alleles could form before the temporal window for homology-directed repair.

Since multiplexing of gRNAs can best be accomplished by expressing them from a single compact promoter, we modified our drive system to include a transfer RNA (tRNA) that must be spliced out of the gRNA gene to generate an active gRNA. By including additional tRNAs between gRNAs, several gRNAs can be expressed together with this system (31). We found that drive/wild-type heterozygote females had a drive conversion efficiency of 82% (95% CI, 78 to 86%) in females and 65% (95% CI, 62 to 70%) in males for the one-gRNA drive with the tRNA (data S2). This indicates that the tRNA system functions correctly in homing drives without apparent loss of efficiency, allowing its use in multiplexed gRNA experiments.

We next constructed a drive to determine the effects of reduced homology between the cleaved wild-type chromosome and the drive allele. We used a single gRNA as above with the tRNA system but with the right homology arm realigned to a hypothetical second gRNA cut site (Fig. 2). Thus, the first 114 nucleotides to the right of the cut site would not be homologous to any DNA around the drive allele. Drive conversion rates for females were only 84% (95% CI, 77 to 90%) of the rate of the one-gRNA drive that had full homology around the cut site, while the rate for males was 89% (95% CI, 83 to 96%) of the full homology drive (data S3). This indicates that a multiple-gRNA drive would indeed exhibit lower conversion efficiency when cleavage does not take place at both ends.

Blue shows gRNA target sites, and black shows regions of DNA that have no homology to the drive allele. Highly active gRNAs are shown by a red lightning bolt, and gRNAs with low activity are shown with an orange line icon.

To assess the effects of Cas9 activity saturation, we examined three constructs containing Cas9 with either zero, one, or four gRNAs targeting a genomic region located between two genes and downstream of both to minimize potential interference with native genes. Mutations resulting from the repair of cleavage events in this area are thereby unlikely to affect an individuals fitness. These constructs were placed at the same genomic site as the synthetic target site constructs. Individuals with these constructs were crossed to those carrying the split drive targeting yellow developed previously (15) to generate individuals heterozygous for both a Cas9 element and a split-drive element. These individuals all had a single gRNA targeting yellow and a variable number of gRNAs that target a region where sequence changes produce no phenotype. The embryo resistance allele formation rates in individuals with zero, one, or four gRNAs that were not targeting yellow in the Cas9 element were 83% (95% CI, 80 to 87%), 72% (95% CI, 68 to 77%), and 65% (95% CI, 60 to 70%), respectively (data S4). The differences between the construct with zero additional gRNA elements and the others were statistically significant (P < 0.0001 in both cases, Fishers exact test), although the difference between the constructs with one and four additional gRNAs did not quite reach statistical significance (P = 0.06, Fishers exact test). The amount of the gRNA targeting yellow was constant in these drives. However, the rate at which yellow was cleaved decreased as the number of other gRNAs increased. This is consistent with the hypothesis that saturation of Cas9 activity reduces the cleavage rates at individual gRNA target sites when the total number of gRNAs is increased.

Nevertheless, Cas9 does not necessarily become fully saturated with a single gRNA. The total cleavage rate could potentially somewhat increase if additional gRNAs are provided, although it would likely plateau rapidly. When heterozygotes for the split drive targeting yellow (15) and the standard drive targeting yellow (12) (which had one copy of Cas9 and two copies of the gRNA gene) were crossed to w1118 males, the rate of embryo resistance allele formation and mosaicism was somewhat higher than for standard drive/resistance allele heterozygotes with one copy of Cas9 and only one gRNA gene (P = 0.0036, Fishers exact test) (data S5).

To assess the performance of drives with multiple gRNAs, we created several additional constructs targeting EGFP, but with two, three, or four gRNAs (Fig. 2). The left target site for each of these was the same as for the one-gRNA synthetic target site drives, and the homologous ends of all of these drives matched the left and right gRNA target sites. However, we found that of the four gRNAs used, only the first and the third had high cleavage activity, as indicated by sequencing of embryo resistance alleles (table S1). Although germline cleavage activity was likely somewhat higher than in the embryo for these gRNAs, their low activity undoubtedly reduced drive performance. Nevertheless, we found that the overall performance of these drives was consistent with the performance predicted by our model that included the effects of timing, repair fidelity, and Cas9 activity saturation (Fig. 2). The results show that adding additional gRNAs does not exponentially increase the efficiency of homing drives.

Specifically, we constructed two different two-gRNA drives. One of these had two closely spaced gRNA targets (36 nucleotides apart) and showed a drive conversion efficiency of 78% (95% CI, 74 to 83%) in females and 62% (95% CI, 56 to 67%) in males (data S6). This was slightly higher than the second drive where the two gRNAs were more widely spaced (114 nucleotides apart), which demonstrated a drive conversion efficiency of 74% (95% CI, 70 to 79%) in females and 60% (95% CI, 55 to 64%) in males (data S7). Because the second gRNA has low activity in each of these drives, the small difference in the performance between them could possibly be accounted for by the lower repair fidelity in the drive with more widely spaced gRNAs (Fig. 2) when only one target site is cut. The drive with three gRNAs was similar to the two-gRNA drive with widely spaced gRNAs, with the addition of a third active gRNA in between the two target sites of the two-gRNA drive. This likely increased the overall cleavage rate due to the higher proportion of active gRNAs and allowed for greater repair fidelity on the right end, since cleavage in this system usually takes place at the left and middle gRNA targets, instead of often only at the left gRNA target. Thus, this construct showed an improved drive conversion efficiency of 80% (95% CI, 77 to 84%) in females, although male drive conversion efficiency apparently remained at 60% (95% CI, 56 to 64%). A final construct added an additional gRNA between the left and middle gRNAs (the same gRNA that the closely spaced two-gRNA construct included). However, since this gRNA had low activity, overall drive performance may have been negatively affected by saturation of Cas9 by gRNAs, resulting in a reduced drive conversion efficiency of 73% (95% CI, 69 to 76%) in females, although male drive conversion efficiency appears to have improved to 65% (95% CI, 61 to 68%) (possibly due to an underestimation of conversion efficiency for males with the three-gRNA construct).

To further refine our model, we next incorporated distinct phases for homing drive dynamics in the germline (Fig. 3). In this model, we assume that first, early germline resistance alleles form, followed by a homology-directed repair phase, and then a late germline resistance allele formation phase. In the embryos of mothers with at least one drive allele, maternally deposited Cas9 and gRNA can result in the formation of additional resistance alleles. During this process, deletions can occur if cleavage occurs nearly simultaneously at different cut sites. If a second site is cleaved before a first cleavage has been repaired, then the section of DNA between the two sites is excised when the gap is closed by end joining repair.

First, wild-type gRNA target sites can be cleaved in the early germline, forming resistance alleles. Next, cleavage occurs at a high rate in the homology-directed repair phase. Usually, this results in successful conversion to a drive allele. However, if homology-directed repair fails to occur, then end joining can form resistance alleles. Incomplete homology-directed repair can also convert the entire allele to a resistance allele, ignoring individual target sites. Next, another resistance allele formation phase converts most remaining wild-type sites into resistance sequences. Meiosis and fertilization take place, and then, if the female parent had at least one drive allele, a final phase of resistance allele formation takes place in the early embryo.

We additionally model reduced repair fidelity from imperfect homology around the cut sites, Cas9 activity saturation, and variance in the activity level of individual gRNAs. See the Supplementary Results for a detailed treatment of these model components and estimation of parameters based on our experiments. Models with repair fidelity or Cas9 activity saturation alone did not produce much deviance from our basic model with timing (Fig. 4). However, a model that includes both repair fidelity and Cas9 activity saturation demonstrated fundamentally changed dynamics. We found that there was a synergistic effect between the factor of the reduced cut rate per site caused by Cas9 activity saturation and the factor of reduced repair fidelity when the outermost target sites are not cut. Because of this, we find the emergence of an optimal number of gRNAs to maximize drive conversion efficiency (Fig. 4), which decreases rapidly when additional gRNAs are added. The additional modeling of gRNA activity variance had only a small negative effect on drive conversion performance (Fig. 4).

Five million offspring were generated from crosses between drive/wild-type heterozygotes and wild-type individuals for each model and number of gRNAs. The rate at which wild-type alleles are converted to drive alleles in the germline of drive/wild-type individuals is shown.

With parameters simulating an efficient A. gambiae construct, the optimal number of gRNAs in this model is two, although drives with three gRNAs have nearly as good conversion efficiency (Fig. 4). Thus, not only do further increases in the number of gRNAs fail to provide substantial benefits, they actually result in substantial reductions in drive efficiency. However, note that the optimal number of gRNAs for overall performance may be somewhat greater than the optimal number for drive conversion efficiency, as detailed below.

Resistance alleles can either preserve or disrupt the function of a target gene. The latter are expected to be more common due to frameshift mutations or other disruptions to the target sequence but should usually be less detrimental to drive performance. In our model, we assume that resistance sequences preserving the function of the target gene form in 10% of cases (12, 13), although this could be substantially reduced by targeting conserved sequences (13, 16, 21). We further assume that if even a single resistance sequence that disrupts the function of the target gene is present, the target gene is rendered nonfunctional. Any deletion due to simultaneous cleavage followed by end joining repair is also assumed to disrupt the target gene. One major advantage of multiple-gRNA drives is therefore that complete resistance alleles that preserve the function of the target gene should become exponentially less common as the number of gRNAs increases (Fig. 5, black line).

Five million offspring were generated from crosses between drive/wild-type heterozygotes and wild-type individuals for each number of gRNAs and each level of probability that incomplete homology-directed repair results in the formation of resistance alleles that preserve the function of the target gene. The formation rate of resistance alleles that preserve the function of the target gene is shown. No such resistance alleles were formed in systems with at least four gRNAs, except in drives where incomplete homology-directed repair was possible.

However, certain types of gene drives are vulnerable to incomplete homology-directed repair as another mechanism for forming resistance alleles that preserve the function of the target gene. These drives target a critical gene such that individuals are rendered nonviable if one (haplolethal) or both (recessive lethal) alleles are disrupted. The drives contain a recoded sequence of the targeted gene that is immune to cleavage by the drives gRNAs. If homology-directed repair copies the recoded portion of the drive, a complete resistance allele that preserves the function of the target gene is formed, regardless of the number of gRNA targets in the system. For modification drives, this is not an issue if the payload is also copied. These events are likely to be even more rare than copying of only the rescue element (because the rescue element is often located at the end of a drive). It is similarly unlikely for the rescue and drive elements to be copied without the payload. Thus, the only outcome of incomplete homology-directed repair that we model is full resistance formation, either disrupting or, more rarely, preserving the function of the target gene. A more detailed discussion of this mechanism is provided in the Supplementary Results covering incomplete homology-directed repair. With incomplete homology-directed repair as the last element in our full model, we find that there is an optimal number of gRNAs for this family of drives for minimizing resistance alleles that preserve the function of the target gene. This number is usually three, but it is somewhat higher when the rate of incomplete homology-directed repair copying the recoded region is very low (Fig. 5).

With our full model in place, we consider the performance of several types of drives. The first of these is the standard homing drive. This drive accomplishes its goal by carrying an engineered payload and targets a neutral locus. Consequently, there is no effect from disrupting the target, and all resistance alleles are treated the same. The next is a suppression drive targeting a recessive female fertility gene (21). In this drive, females are rendered sterile unless they have at least one wild-type allele or a resistance allele that preserves the function of the target gene. The dynamics of this drive result in complete population suppression when it is successful. We also consider approaches for population modification that target a haplolethal or recessive lethal gene, where the drive has a recoded sequence of the gene that is immune to gRNA cleavage (16). In the haplolethal approach, any individual with a resistance allele that disrupts the target gene is nonviable, removing these alleles from the population. In the recessive lethal approach, an individual is only nonviable if it has two such resistance alleles. Last, we consider a population modification drive that targets a gene of interest, such as a gene required for malaria transmission in Anopheles (27, 28). Rather than carrying a payload, this drives purpose is to disrupt its target in a manner similar to that of the suppression drive.

We found that the optimal number of gRNAs for the population modification drives to achieve a maximum drive frequency was three, although drives with two gRNAs were nearly as efficient (Fig. 6A). The haplolethal drive reached nearly 100% frequency when modeled with two or more gRNAs (Fig. 6A) due to rapid removal of resistance alleles. The recessive lethal drive is slower at removing resistance alleles when they form at low rates, so it reached a lower frequency (Fig. 6A). However, the haplolethal drive also removes drive alleles when they are present in the same individual as a resistance allele that disrupts the function of the target gene. Thus, this system spreads somewhat more slowly than other types of population modification drives, although not as slowly as the population suppression homing drive (Fig. 6B). Of particular interest, gRNAs beyond two reduce drive conversion efficiency, which results in a slower spread of the drive (Fig. 6B). However, having multiple gRNAs is essential for reducing the formation rate of resistance alleles that preserve the function of the target gene (Fig. 5), which would otherwise outcompete drive alleles over time (Fig. 6C).

Drive/wild-type heterozygotes were released into a population of 100,000 individuals at an initial frequency of 1%. The simulation was then conducted for 100 generations using the full model. The displayed results are the average from 20 simulations for each type of drive and number of gRNAs. (A) The maximum drive allele frequency reached at any time in the simulations. Note that the standard drive and gene disruption drive have nearly identical values. (B) Number of generations needed for the drive to reach at least 50% total allele frequency. Note that the suppression drive is only shown in (B). (C) Final frequency of resistance alleles after 100 generations. The displayed values are only for resistance alleles that preserve the function of the target gene. No resistance alleles were present in the standard drive and gene disruption drive when at least four gRNAs were present. (D) Final effector allele frequency in the population after 100 generations. This was the drive allele for most drive types, but for the gene disruption drive, it includes resistance alleles that disrupt the function of the target gene as well.

Overall, having three gRNAs is usually optimal for population modification drives to attain maximum drive frequency after 100 generations (Fig. 6D). However, for the gene disruption homing drive, the optimal number of gRNAs for maximizing the frequency of effector alleles was four, five, or six (Fig. 6D). This is because effector alleles for this drive also include resistance alleles that disrupt the function of the target gene. In addition, this type of drive is not substantially impaired by incomplete homology-directed repair. This means that gene disruption drive can make efficient use of a higher number of gRNAs. Drives modeled with somewhat reduced performance based on our Drosophila experiments in this study (albeit with slightly lowered embryo resistance allele formation rates to represent an improved promoter) showed similar patterns, but with the optimal number of gRNAs increased by one for each drive (fig. S5 and see the Supplementary Results).

Suppression-type homing drives are particularly prone to failure if the resistance allele formation rate is high or if the drive conversion efficiency is too low. When examining the rate at which the drive was successful in completely suppressing the population (Fig. 7A), our high-performance drives with default parameters were usually successful, so long as there were sufficiently many gRNAs. However, drives with somewhat reduced performance (see the Supplementary Results) were less able to achieve successful suppression, regardless of the number of gRNAs. As with the default parameters, low numbers of gRNAs resulted in formation of resistance alleles that preserved the function of the target gene, which were able to quickly reach fixation in the population and prevent suppression (Fig. 7B). With an intermediate number of gRNAs, complete population suppression still usually occurred (Fig. 7C), but when the number of gRNAs was high, the rate of complete suppression declined. This is because with a high number of gRNAs, the drive suffered from reduced conversion efficiency and lacked the power to completely suppress the population (Fig. 7D). As the number of gRNAs is increased beyond two to three, the genetic load imposed by the drive at its final equilibrium (in the absence of resistance alleles that preserve the function of the target gene) is substantially reduced (fig. S6), preventing the drive from inducing complete suppression if the population growth rate at low densities is sufficiently high. With a choice of target sites with reduced formation of resistance sequences that preserve the function of the target gene, complete suppression becomes more likely, and the optimal number of gRNAs is reduced (fig. S7).

Drive/wild-type heterozygotes with a suppression drive were released into a population of 100,000 individuals at an initial frequency of 1%. The simulation was then conducted for 100 generations. (A) The displayed results are the average from 20 simulations for each type of drive and number of gRNAs. The fraction of simulations that resulted in complete suppression is shown. The full model was used. The default system based on the Anopheles parameters used an early germline cleavage rate of 2%, a homology-directed repair phase cleavage rate of 98%, and an embryo cleavage rate of 5%. For the reduced efficiency drive model, these parameters were changed to 5, 92, and 10%, respectively. The low efficiency drive model changed these parameters to 8, 90, and 15%, respectively. Allele frequency and population size trajectories are shown for individual simulations using the reduced efficiency model with (B) 2, (C) 4, and (D) 10 gRNAs. r1 refers to resistance alleles that preserve the function of the target gene, and r2 refers to resistance alleles that disrupt the function of the target gene.

Our study shows that homing drives likely have an optimal number of gRNAs that maximize drive efficiency while minimizing the formation of resistance alleles that preserve the function of the target gene. This result emerged naturally from a model that incorporated specific time steps for cleavage and repair, Cas9 activity saturation, and reduced repair fidelity when homology ends around the cut sites fail to line up perfectly. Even with a more basic model that differs from the model only by allowing a narrow timing window for homology-directed repair, we are able to reject the notion that homing gene drives can be made arbitrarily efficient by having a sufficiently high number of gRNAs. Overall, we showed that while multiple gRNAs are useful for improving drive efficiency and reducing resistance, these performance gains are far smaller than those predicted by simple models with sequential cutting and repair (2224) or even models that include simultaneous cutting (22). This new model is consistent with our experimental results in this study, as well as previous work that observed smaller improvements from multiple gRNAs than predicted (13) or even marked declines in performance (9). Our model also takes germline cleavage timing into account, which is consistent with resistance allele sequencing in previous experimental studies (10, 12, 13).

While our model represents a step forward in our understanding of how multiplexed gRNAs affect homing drive efficiency, further improvements are needed to be able to more accurately predict homing drive performance. Earlier work indicated that the window for homology-directed repair is narrow, with only resistance alleles forming before and afterward (10, 12, 13). A better understanding of this window, the rate of successful homology-directed repair, and the proportion of resistance alleles formed before, during, and after this window would allow for improvements to our model. Homology of DNA on either side of a cut site is well known to be critical for the fidelity of homology-directed repair, and we showed that it indeed influences drive conversion efficiency. Last, Cas9 cleavage activity always reaches a maximum as more gRNAs are added, although details of this have not yet been thoroughly quantified. It is likely that for many gRNA promoters, a maximum cut rate would be reached quickly, thus reducing the cleavage rates at individual gRNA target sites as the total number is increased. Future studies could investigate how this saturation occurs and enable refinement of the quantitative model. In particular, the rate of resistance alleles formed due to incomplete homology-directed repair could be better quantified, with particular attention paid to the rate at which any recoded region is fully copied, thereby forming a resistance allele that preserves the function of the target gene. Last, variance in the activity level of gRNAs is well known, and we also observed this in our multiple gRNA homing drives in this study. These activity levels could potentially be predicted (32), but experimental assessment will likely remain necessary in the foreseeable future.

In our model, we also assumed that each gRNA cut site independently had the same chance of forming a resistance sequence that disrupts the function of the target gene. Thus, gRNA target sites would be best located close together to maximize repair fidelity. In practice, frameshifts between gRNA cut sites, but with restored frame after the last mutated site, may be insufficient to disrupt the function of the target gene. Thus, a good practice to minimize the formation of resistance alleles that preserve the function of the target gene would be to target conserved or important regions less tolerant of mutations, and perhaps to space gRNAs far enough apart, despite the cost to drive conversion efficiency, to ensure that a frameshift between any two gRNA sites disrupts the gene. At minimum, gRNAs should be placed far enough apart to prevent mutations at one site from converting an adjacent target site into a resistance allele.

Our models allowed us to gain insights about the relative strengths and weaknesses of the different types of homing gene drives. Standard drives lack any particular mechanism for removing resistance alleles (they need not even target a specific gene), which means that a successful drive of this nature requires a high drive efficiency, very low resistance allele formation rates, and low fitness costs to persist long enough to provide substantial benefits. The optimal number of gRNAs for these drives is likely low, perhaps two or three for a highly efficient system.

By contrast, drives that target haplolethal or recessive lethal genes can effectively remove resistance alleles that disrupt the function of the target gene and, thus, tolerate substantially higher overall rates of resistance allele formation. These drives are not expected to lose much efficiency with larger numbers of gRNAs, because although drive conversion efficiency is reduced, the drives also operate by toxin-antidote principles (25, 26), enabling removal of wild-type alleles and an accompanying relative increase in drive allele frequency even without drive conversion. However, we hypothesize that with reduced homology around the cut sites, incomplete homology-directed repair becomes more likely. This results in an optimal level of gRNAs that minimizes the formation of resistance alleles that preserve the function of the target gene due to incomplete homology-directed repair and end joining mechanisms. It is unclear how often incomplete homology-directed repair occurs, but it is likely that the optimal number of gRNAs for these drives is perhaps three or four. However, the rate of incomplete homology-directed repair could perhaps be minimized if the drive is located in an intron (possibly a synthetic intron), with essential recoded regions on either side of the intron. A system of this nature would only form a resistance allele that preserves the function of the target gene if incomplete homology-directed repair were to occur on both sides of the drive. This would allow for efficient use of a greater number of gRNAs. Improvements of this nature may not be necessary, however, if the rate of resistance allele formation that preserves the target function is substantially less than the rate at which payload genes are inactivated by mutations that occur during homology-directed repair (106 per nucleotide), which is approximately 1000-fold greater than the rate by DNA replication. If such a rate would preclude effective deployment of a homing drive, then toxin-antidote systems (25, 26) that rely only on DNA replication for copying of payload genes may be more suitable.

A gene disruption homing drive for population modification could potentially avoid both the need for a recoded region and inactivation of payload genes by targeting an endogenous gene. In this case, the end goal would be to disrupt this gene either by the presence of the drive or by formation of resistance alleles, rather than spreading a specific payload, and the formation of resistance alleles that disrupt the target gene may actually be beneficial due to their reduced fitness cost compared to the drive. For such a drive, the optimal number of gRNAs would be the minimum number necessary to prevent the formation of resistance alleles that preserve the function of the target gene, perhaps four to eight, depending on population size, target site, and drive performance.

A drive designed for population suppression has similar considerations, but with a narrower window for success. This is because any formation of complete resistance alleles that preserve the function of the target gene would likely result in rapid failure of the drive. In addition, if drive conversion efficiency is insufficient, then the drive may lack the power to completely suppress the population, at least within a reasonable timeframe. Thus, a narrower range of five to seven gRNAs would likely be optimal for such a drive. For all of these drive types, if the rate of resistance allele formation that preserves the function of the target gene is lower than in our models (such as by targeting a sequence that is highly intolerant of mutations (21)), then the optimal number of gRNAs is somewhat reduced.

Overall, we conclude that the total number of gRNA should be kept relatively low to achieve maximum effectiveness of multiple-gRNA drives: at least two, but well under a dozen, with the exact number depending on the type of drive and other performance characteristics. The gRNA target sites should also be placed as close together as possible while still far enough apart to prevent mutations at one target site from affecting adjacent sites. While our results suggest that multiplexing of gRNAs alone is unlikely to enable the development of highly effective homing drives, we expect that this approach will still be a critical component of any successful drive, especially when combined with additional strategies.

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Computational and experimental performance of CRISPR homing gene drive strategies with multiplexed gRNAs - Science Advances

For the first time, doctors have used a CRISPR gene editing therapy in an attempt to fix broken genes within the body, marking another step forward for a technology that promises to change how some inherited diseases are treated.

Clinicians at Oregon Health and Science University recently injected the therapy, developed by biotech Editas Medicine and partner Allergan, into the eye of a patient with a type of severe blindness, the companies confirmed Wednesday.

A study last year tested another CRISPR medicine in stem cells extracted from patients' blood, while a third trial previously used a different type of gene editing technology called zinc finger nucleases inside the body. But the patient recently given Editas and Allergan's therapy is the first to be treated using a CRISPR therapy that works in vivo.

The eye disease the companies hope to correct, called Leber cogenital amaurosis, is caused by mutations in any of at least a dozen genes. Editas and Allergan are focusing on just one particular type, known as LCA10. Between 2,000 and 5,000 patients in the U.S. and Europe have it, according to the companies.

"Half of the patients who have this disease are born essentially with light perception vision. They can tell that the room is dark or light," said Mark Pennesi, an associate professor of ophthalmology who is leading OHSU's involvement in the study, in an interview.

"The other half start at legal blindness and then will degrade over the first two decades of life."

Pennesi and his colleagues hope Editas and Allergan's medicine could restore vision by deleting the mutation that prevents the eye from making a protein critical to light-detecting cells.

If that protein is made again, the damaged segment of those photoreceptors should be able to regenerate, said Charles Albright, chief scientific officer at Editas, in an interview last month.

Editas and Allergan plan to enroll 18 adults and children into the study, which is currently being conducted at OHSU as well as centers in Miami, Boston and Ann Arbor, Michigan.

The initial focus will be on safety, as researchers gauge whether the CRISPR medicine being tested causes any side effects or toxicities. Should all go well with the first few adults given a low dose, Editas and Allergan will test four higher doses and potentially try the therapy in children.

Enrolling patients into the study, dubbed BRILLIANCE, has taken longer than the companies first expected when they opened the trial last July.

"Getting patients enrolled and recruiting has taken longer than planned," said Albright, noting there were prospective study participants who came in but ultimately weren't eligible for dosing.

Moving forward, Albright said enrollment should proceed more smoothly.

Whether the treatment helps improve vision will be measured using eye charts and a "mobility maze" similar to one used by Spark Therapeutics for its gene therapy Luxturna, approved in late 2017 for a different type of inherited blindness.

Luxturna works not by editing DNA, but rather by inserting a functional copy of a defective gene directly into the eye. That approach wasn't possible with LCA10, Pennesi said, because the gene in question is too large to fit into the inactivated viruses companies are using as delivery vehicles.

Editing DNA holds potential risks, however the greatest being that the CRISPR therapy inadvertently cuts DNA in places the companies and researchers don't intend and makes irreversible changes.

As with all firsts, the long-term effects of gene editing aren't known either, although Albright noted that photoreceptor cells in the eye no longer divide, potentially making the results of Editas and Allergan's therapy more predictable.

While Editas and Allergan are first to the milestone of in vivo CRISPR editing, the field around them is quickly advancing.

CRISPR Therapeutics and Vertex, which are running the study that used a CRISPR therapy on extracted stem cells, already have initial data, while rival Intellia Therapeutics plans to begin this year a study of in vivo CRISPR editing in a rare disease known as transtheyretin amyloidosis.

Other, newer companies, meanwhile, are working to move past CRISPR and into more specific types of gene editing. One, the Cambridge, Massachusetts-based Beam Therapeutics, recently raised $207 million on the promise of its base-editing platform.

But the studies run by CRISPR Therapeutics and Editas, being the first in their respective settings, will be watched close.

"These are setting precedent," said Albright. "You're going to be seeing a lot more gene editing."

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In a CRISPR first, Editas therapy used to fix genes in the body - BioPharma Dive

Ebola. Sickle cell disease. Spinal muscular atrophy. Cystic fibrosis.

Everyone agrees the void is a problem, but theres little consensus on how to tackle it and theres no panacea to speak of.

Behind each disease was a medical breakthrough that Francis Collins highlighted at the congressional hearing on the presidents 2021 NIH budget request, a yearly opportunity to update lawmakers on his agencys progress and priorities. Thanks to three decades of research that dates in part back to his own NIH-backed work at the University of Michigan, for instance, the US has ushered in its first triple therapy for cystic fibrosis last year.

These are dramatic times for NIH research, the director concluded.

Bolstering the burst in new scientific discovery and therapeutic development has been an impressive growth in NIH funding. President Donald Trump may be proposing to cut its budget down 7% next year, but over the past five years it has increased by $11.6 billion, or 39%, according to Rep Rosa DeLauro, chair of the House Appropriations subcommittee on Labor, Health & Human Services and Education. That has translated to a $8 billion boost to the total amount of grants awarded between 2014 and 2019, per NIH disclosure.

The steady increases you have provided have brought new life to biomedical research and built the foundation for us to take on new and unexpected challenges, Collins said, challenges like the one thats on everyones mind right now: the global coronavirus outbreak.

What does this new life look like on the ground? Endpoints News spoke to researchers, administrators and advocates, who pointed to different metrics that either measure output or the environment that scientists find themselves working in. The conversations suggest while the increases which followed years of stagnation did pump more resources into translatioal research, they didnt quite solve the challenges basic science still faces.

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Tackling inherited blindness, Editas and Allergan use CRISPR for the first time in the human body - Endpoints News

AGN-151587 (EDIT-101) is the firstin vivoCRISPR medicine to be administered to patients

Additional patient enrollment to the BRILLIANCE Clinical Trial is ongoing

DUBLIN, Ireland and CAMBRIDGE, Mass., March 04, 2020 (GLOBE NEWSWIRE) -- Allergan plc (NYSE: AGN), a leading global pharmaceutical company, and Editas Medicine, Inc. (Nasdaq: EDIT), a leading genome editing company, today announced the treatment of the first patient in the BRILLIANCE clinical trial of AGN-151587 (EDIT-101) at Oregon Health & Science University (OHSU) Casey Eye Institute, a world-recognized academic eye center.

AGN-151587 (EDIT-101) is an experimental medicine delivered via sub-retinal injection under development for the treatment of Leber congenital amaurosis 10 (LCA10), an inherited form of blindness caused by mutations in the centrosomal protein 290 (CEP290) gene. The BRILLIANCE clinical trial is a Phase 1/2 study to evaluate AGN-151587 for the treatment of patients diagnosed with LCA10 and is the worlds first human study of an in vivo, or inside the body, CRISPR genome editing medicine. The trial will assess the safety, tolerability, and efficacy of AGN-151587 in approximately 18 patients with LCA10.

This dosing is a truly historic event for science, for medicine, and most importantly for people living with this eye disease, said Cynthia Collins, President and CEO, Editas Medicine. The first patient dosed in the BRILLIANCE clinical trial marks a significant milestone toward delivering on the promise and potential of CRISPR medicines to durably treat devastating diseases such as LCA10. We look forward to sharing future updates from this clinical trial and our ocular program.

Currently patients living with LCA10 have no approved treatment options. For years, Allergan has had an unwaveringcommitmentto advancingeyecare treatments. With the first patient treated in this historic clinical trial, we mark a significant step in advancing the AGN-151587 clinical program and move closer to our goal of developing a game-changing medicine for LCA10 patients, said Brent Saunders, Chairman and CEO, Allergan.

Our first treatment in this clinical trial is an important step toward bringing new and promising treatments to patients with disease-causing gene mutations. OHSU is honored to be involved in this effort to address previously untreatable diseases such as Leber congenital amaurosis 10, said Mark Pennesi, M.D., Ph.D., Associate Professor of Ophthalmology, Kenneth C. Swan Endowed Professor, Division Chief, Paul H. Casey Ophthalmic Genetics, Casey Eye Institute, Oregon Health & Science University, Principal Investigator and enrolling physician of the first patient treated with AGN-151587.

Eric A. Pierce, M.D., Ph.D., Director of the Inherited Retinal Disorders Service and Director of the Ocular Genomics Institute at Massachusetts Eye and Ear, and the William F. Chatlos Professor of Ophthalmology at Harvard Medical School, and a Principal Investigator for the BRILLIANCE clinical trial also commented, We have a long history at Massachusetts Eye and Ear of helping develop life-changing medicines for our patients, and we are thrilled to be a leader in the development of a CRISPR-based experimental medicine to treat CEP290-associated retinal disease with Allergan and Editas.

About the BRILLIANCE Phase 1/2 Clinical Trial of AGN-151587 (EDIT-101)The BRILLIANCE Phase 1/2 clinical trial of AGN-151587 (EDIT-101) for the treatment of Leber congenital amaurosis 10 (LCA10) will assess the safety, tolerability, and efficacy of AGN-151587 in approximately 18 patients with this disorder. Up to five cohorts of patients across three dose levels will be enrolled in this open label, multi-center, clinical trial. Both adult and pediatric patients (3 17 years old) with a range of baseline visual acuity assessments are eligible for enrollment. Patients will receive a single administration of AGN-151587 via subretinal injection in one eye. Additional details are available on http://www.clinicaltrials.gov (NCT#03872479).

About AGN-151587 (EDIT-101)AGN-151587 (EDIT-101) is a CRISPR-based experimental medicine under investigation for the treatment of Leber congenital amaurosis 10 (LCA10). AGN-151587 is administered via a subretinal injection to deliver the gene editing machinery directly to photoreceptor cells.

About Leber Congenital AmaurosisLeber congenital amaurosis, or LCA, is a group of inherited retinal degenerative disorders caused by mutations in at least 18 different genes.It is the most common cause of inherited childhood blindness, with an incidence of two to three per 100,000 live births worldwide.Symptoms of LCA appear within the first years of life, resulting in significant vision loss and potentially blindness.The most common form of the disease, LCA10, is a monogenic disorder caused by mutations in the CEP290 gene and is the cause of disease in approximately 2030 percent of all LCA patients.

About the Editas Medicine-Allergan AllianceIn March 2017, Editas Medicine and Allergan Pharmaceuticals International Limited (Allergan) entered a strategic alliance and option agreement under which Allergan received exclusive access and the option to license up to five of Editas Medicines genome editing programs for ocular diseases, including AGN-151587 (EDIT-101).Under the terms of the agreement, Allergan is responsible for development and commercialization of optioned products, subject to Editas Medicines option to co-develop and share equally in the profits and losses of two optioned products in the United States. Editas Medicine is also eligible to receive development and commercial milestones, as well as royalty payments on a per-program basis.The agreement covers a range of first-in-class ocular programs targeting serious, vision-threatening diseases based on Editas Medicines unparalleled CRISPR genome editing platform, including CRISPR/Cas9 and CRISPR/Cpf1 (also known as Cas12a). In August 2018, Allergan exercised its option to develop and commercialize AGN-151587 globally for the treatment of LCA10. Additionally, Editas Medicine exercised its option to co-develop and share equally in the profits and losses from AGN-151587 in the United States.

About Allergan plcAllergan plc (NYSE: AGN), headquartered in Dublin, Ireland, is a global pharmaceutical leader focused on developing, manufacturing and commercializing branded pharmaceutical, device, biologic, surgical and regenerative medicine products for patients around the world. Allergan markets a portfolio of leading brands and best-in-class products primarily focused on four key therapeutic areas including medical aesthetics, eye care, central nervous system and gastroenterology. As part of its approach to delivering innovation for better patient care, Allergan has built one of the broadest pharmaceutical and device research and development pipelines in the industry.

With colleagues and commercial operations located in approximately 100 countries, Allergan is committed to working with physicians, healthcare providers and patients to deliver innovative and meaningful treatments that help people around the world live longer, healthier lives every day.

For more information, visit Allergans website atwww.Allergan.com.

About Editas Medicine As a leading genome editing company,Editas Medicineis focused on translating the power and potential of the CRISPR/Cas9 and CRISPR/Cas12a (also known as Cpf1) genome editing systems into a robust pipeline of treatments for people living with serious diseases around the world.Editas Medicineaims to discover, develop, manufacture, and commercialize transformative, durable, precision genomic medicines for a broad class of diseases. For the latest information and scientific presentations, please visit http://www.editasmedicine.com.

Allergan Forward-Looking StatementsStatements contained in this press release that refer to future events or other non-historical facts are forward-looking statements that reflect Allergans current perspective on existing trends and information as of the date of this release. Actual results may differ materially from Allergans current expectations depending upon a number of factors affecting Allergans business. These factors include, among others, the difficulty of predicting the timing or outcome of FDA approvals or actions, if any; the impact of competitive products and pricing; market acceptance of and continued demand for Allergans products; the impact of uncertainty around timing of generic entry related to key products, including RESTASIS, on our financial results; risks associated with divestitures, acquisitions, mergers and joint ventures; risks related to impairments; uncertainty associated with financial projections, projected cost reductions, projected debt reduction, projected synergies, restructurings, increased costs, and adverse tax consequences; difficulties or delays in manufacturing; and other risks and uncertainties detailed in Allergans periodic public filings with the Securities and Exchange Commission, including but not limited to Allergan's Annual Report on Form 10-K for the year ended December 31, 2019. Except as expressly required by law, Allergan disclaims any intent or obligation to update these forward-looking statements.

Editas Medicine Forward-Looking StatementsThis press release contains forward-looking statements and information within the meaning of The Private Securities Litigation Reform Act of 1995. The words anticipate, believe, continue, could, estimate, expect, intend, may, plan, potential, predict, project, target, should, would, and similar expressions are intended to identify forward-looking statements, although not all forward-looking statements contain these identifying words. Forward-looking statements in this press release include statements regarding the Companies plans with respect to the Phase 1/2 clinical trial for AGN-151587 (EDIT-101).Editas Medicine may not actually achieve the plans, intentions, or expectations disclosed in these forward-looking statements, and you should not place undue reliance on these forward-looking statements. Actual results or events could differ materially from the plans, intentions and expectations disclosed in these forward-looking statements as a result of various factors, including: uncertainties inherent in the initiation and completion of preclinical studies and clinical trials and clinical development of Editas Medicines product candidates; availability and timing of results from preclinical studies and clinical trials; whether interim results from a clinical trial will be predictive of the final results of the trial or the results of future trials; expectations for regulatory approvals to conduct trials or to market products and availability of funding sufficient for Editas Medicines foreseeable and unforeseeable operating expenses and capital expenditure requirements. These and other risks are described in greater detail under the caption Risk Factors included in Editas Medicines most recent Annual Report on Form 10-K, which is on file with the Securities and Exchange Commission, and in other filings that Editas Medicine may make with the Securities and Exchange Commission in the future. Any forward-looking statements contained in this press release speak only as of the date hereof, and Editas Medicine expressly disclaims any obligation to update any forward-looking statements, whether because of new information, future events or otherwise.

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Allergan and Editas Medicine Announce Dosing of First Patient in Landmark Phase 1/2 Clinical Trial of CRISPR Medicine AGN-151587 (EDIT-101) for the...

THREADNEEDLE ASSET MANAGEMENT LT bought a fresh place in W&T Offshore Inc. (NYSE:WTI). The institutional investor bought 1.9 million shares of the stock in a transaction took place on 12/31/2019. In another most recent transaction, which held on 12/31/2019, HOTCHKIS & WILEY CAPITAL MANAGEM bought approximately 1.5 million shares of W&T Offshore Inc. In a separate transaction which took place on 12/31/2019, the institutional investor, COLUMBIA MANAGEMENT INVESTMENT A bought 1.1 million shares of the companys stock. The total Institutional investors and hedge funds own 63.40% of the companys stock.

In the most recent purchasing and selling session, W&T Offshore Inc. (WTI)s share price decreased by -7.85 percent to ratify at $2.23. A sum of 7588948 shares traded at recent session and its average exchanging volume remained at 2.70M shares. The 52-week price high and low points are important variables to concentrate on when assessing the current and prospective worth of a stock. W&T Offshore Inc. (WTI) shares are taking a pay cut of -68.92% from the high point of 52 weeks and flying high of -5.91% from the low figure of 52 weeks.

W&T Offshore Inc. (WTI) shares reached a high of $2.53 and dropped to a low of $2.20 until finishing in the latest session at $2.48. Traders and investors may also choose to study the ATR or Average True Range when concentrating on technical inventory assessment. Currently at 0.25 is the 14-day ATR for W&T Offshore Inc. (WTI). The highest level of 52-weeks price has $7.18 and $2.37 for 52 weeks lowest level. After the recent changes in the price, the firm captured the enterprise value of $1.05B, with the price to earnings ratio of 1.53. The liquidity ratios which the firm has won as a quick ratio of 0.90, a current ratio of 0.90.

Having a look at past record, were going to look at various forwards or backwards shifting developments regarding WTI. The firms shares fell -21.20 percent in the past five business days and shrunk -45.61 percent in the past thirty business days. In the previous quarter, the stock fell -45.74 percent at some point. The output of the stock decreased -49.32 percent within the six-month closing period, while general annual output lost -55.31 percent. The companys performance is now negative at -59.89% from the beginning of the calendar year.

According to WSJ, W&T Offshore Inc. (WTI) obtained an estimated Overweight proposal from the 3 brokerage firms currently keeping a deep eye on the stock performance as compares to its rivals. 0 equity research analysts rated the shares with a selling strategy, 1 gave a hold approach, 2 gave a purchase tip, 0 gave the firm a overweight advice and 0 put the stock under the underweight category. The average price goal of one year between several banks and credit unions that last year discussed the stock is $7.75.

CRISPR Therapeutics AG (CRSP) shares on Thursdays trading session, jumped 0.28 percent to see the stock exchange hands at $53.41 per unit. Lets a quick look at companys past reported and future predictions of growth using the EPS Growth. EPS growth is a percentage change in standardized earnings per share over the trailing-twelve-month period to the current year-end. The company posted a value of $0.97 as earning-per-share over the last full year, while a chance, will post -$4.97 for the coming year. The current EPS Growth rate for the company during the year is 134.10% and predicted to reach at -10.70% for the coming year. In-depth, if we analyze for the long-term EPS Growth, the out-come was 54.00% for the past five years.

The last trading period has seen CRISPR Therapeutics AG (CRSP) move -27.82% and 59.20% from the stocks 52-week high and 52-week low prices respectively. The daily trading volume for CRISPR Therapeutics AG (NASDAQ:CRSP) over the last session is 1.09 million shares. CRSP has attracted considerable attention from traders and investors, a scenario that has seen its volume jump 1.13% compared to the previous one.

Investors focus on the profitability proportions of the company that how the company performs at profitability side. Return on equity ratio or ROE is a significant indicator for prospective investors as they would like to see just how effectively a business is using their cash to produce net earnings. As a return on equity, CRISPR Therapeutics AG (NASDAQ:CRSP) produces 11.70%. Because it would be easy and highly flexible, ROI measurement is among the most popular investment ratios. Executives could use it to evaluate the levels of performance on acquisitions of capital equipment whereas investors can determine that how the stock investment is better. The ROI entry for CRSPs scenario is at 4.90%. Another main metric of a profitability ratio is the return on assets ratio or ROA that analyses how effectively a business can handle its assets to generate earnings over a duration of time. CRISPR Therapeutics AG (CRSP) generated 9.60% ROA for the trading twelve-month.

Volatility is just a proportion of the anticipated day by day value extendthe range where an informal investor works. Greater instability implies more noteworthy benefit or misfortune. After an ongoing check, CRISPR Therapeutics AG (CRSP) stock is found to be 7.79% volatile for the week, while 6.59% volatility is recorded for the month. The outstanding shares have been calculated 56.28M. Based on a recent bid, its distance from 20 days simple moving average is -1.43%, and its distance from 50 days simple moving average is -6.74% while it has a distance of 5.15% from the 200 days simple moving average.

The Williams Percent Range or Williams %R is a well-known specialized pointer made by Larry Williams to help recognize overbought and oversold circumstances. CRISPR Therapeutics AG (NASDAQ:CRSP)s Williams Percent Range or Williams %R at the time of writing to be seated at 15.77% for 9-Day. It is also calculated for different time spans. Currently for this organization, Williams %R is stood at 48.53% for 14-Day, 54.57% for 20-Day, 72.13% for 50-Day and to be seated 59.61% for 100-Day. Relative Strength Index, or RSI(14), which is a technical analysis gauge, also used to measure momentum on a scale of zero to 100 for overbought and oversold. In the case of CRISPR Therapeutics AG, the RSI reading has hit 47.64 for 14-Day.

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Emerging Stocks to Watch: W&T Offshore Inc. (WTI) and CRISPR Therapeutics AG (CRSP) - BOV News

On Tuesday, shares of CRISPR Therapeutics AG (NASDAQ:CRSP) marked $51.15 per share versus a previous $53.35 closing price. With having a -4.12% loss, an insight into the fundamental values of CRISPR Therapeutics AG, investors would also find a great ally in the technical patterns of the stock movements showed in stock charts. CRSP showed a fall of -16.02% within its YTD performance, with highs and lows between $33.55 $74.00 during the period of 52 weeks, compared to the simple moving average of 0.96% in the period of the last 200 days.

Evercore ISI equity researchers changed the status of CRISPR Therapeutics AG (NASDAQ: CRSP) shares from Outperform to a In-line rating in the report published on February 3rd, 2020. Other analysts, including William Blair, also published their reports on CRSP shares. William Blair repeated the rating from the previous report, marking CRSP under Outperform rating, in the report published on November 19th, 2019. Additionally, CRSP shares got another Outperform rating from Oppenheimer, setting a target price of $65 on the companys shares, according to the report published in November 12th, 2019. On August 1st, 2019, Jefferies Initiated an Buy rating and increased its price target to $64. On the other hand, Canaccord Genuity Initiated the Buy rating for CRSP shares, as published in the report on July 26th, 2019. ROTH Capital seems to be going bullish on the price of CRSP shares, based on the price prediction for CRSP, indicating that the shares will jump to $50, giving the shares Buy rating based on their report from June 10th, 2019. Another Outperform rating came from Evercore ISI.

The present dividend yield for CRSP owners is set at 0, marking the return investors will get regardless of the companys performance in the upcoming period. However, in order for the company to be able to pay its dividends, just like it is the case with CRISPR Therapeutics AG, the company needs to provide a healthy cash flow, currently at the value of 57.57. In addition, the growth of sales from quarter to quarter is recording 66870.40%, hinting the companys progress in the upcoming progress.

In order to gain a clear insight on the performance of CRISPR Therapeutics AG (CRSP) as it may occur in the future, there are more than several well-rounded types of analysis and research techniques, while equity is most certainly one of the more important indicators into the companys growth and performance. In this case, you want to make sure that the return on the present equity of 11.70% is enough for you to make a profit out of your investment. You may also count in the quick ratio of the company, currently set at 17.30 so you would make sure that the company is able to cover the debts it may have, which can be easily seen in annual reports of the company.

Set to affect the volatility of a given stock, the average volume can also be a valuable indicator, while CRSP is currently recording an average of 1.10M in volumes. The volatility of the stock on monthly basis is set at 6.43%, while the weekly volatility levels are marked at 9.16%with 2.53% of gain in the last seven days. Additionally, long-term investors are predicting the target price of $75.96, indicating growth from the present price of $51.15, which can represent yet another valuable research and analysis points that can help you decide whether to invest in CRSP or pass.

CRISPR Therapeutics AG (CRSP) is based in the Switzerland and it represents one of the well-known company operating with Healthcare sector. If you wish to compare CRSP shares with other companies under Electronic Equipment and Consumer Goods, a factor to note is the P/E value of 52.68 for CRISPR Therapeutics AG, while the value can represent an indicator in the future growth of the company in terms of investors expectations. The later value should have a steady growth rate, increasing and growing gradually, which serves the purpose of reliably showcasing the progress of the company. The value 0.97 is supported by the yearly ESP growth of 134.10%.

Besides from looking into the fundamentals, you should also note the number of people inside the company owning the shares, as the values should be in line with the expectations of investors. In that spirit, the present ownership of stocks inside the company is set at 0.30%, which can provide you with an insight of how involved executives are in owning shares of the company. In oppose to the executives share, the institutional ownership counts 53.40% of shares, carrying an equal significance as an indicator of value, as the presence of large investors may signal a strong company.

It appears that more than several institutional investors and hedge funds decided to increase stakes in CRSP in the recent period. That is how Nikko Asset Management Americas, now has an increase position in CRSP by 9.80% in the first quarter, owning 3.05 million shares of CRSP stocks, with the value of $158.42 million after the purchase of an additional 272,139 shares during the last quarter. In the meanwhile, ARK Investment Management LLC also increased their stake in CRSP shares changed 6.27% in the first quarter, which means that the company now owns 2.96 million shares of company, all valued at $153.6 million after the acquisition of additional 174,495 shares during the last quarter.

Federated Global Investment Manag acquired a new position in CRISPR Therapeutics AG during the first quarter, with the value of $65.55 million, and T. Rowe Price Associates, Inc. increased their stake in the companys shares by 59.00% in the first quarter, now owning 411,929 shares valued at $57.67 million after the acquisition of the additional 1.11 million shares during the last quarter. In the end, Credit Suisse Asset Management increased their position by 48.41% during the first quarter, now owning 770792 CRSP shares, now holding the value of $40.04 million in CRSP with the purchase of the additional 760,000 shares during the period of the last quarter. At the present, 53.40% of CRSP shares are in the ownership of institutional investors.

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Here's The Case for and Against CRISPR Therapeutics AG (CRSP) - US Post News

Global CRISPR Technology Market 2020-2025

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Types Covered In This Report:EnzymesKitsgRNALibrariesDesign Tools

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In addition to the factors mentioned above impacting the Global CRISPR Technology Market, this comprehensive research report gauges for decisive conclusions concerning growth factors and determinants, eventually influencing holistic growth and lucrative business models in Global CRISPR Technology Market. Further in the course of the report this research report on Global CRISPR Technology Market identifies notable industry forerunners and their effective business decisions, aligning with market specific factors such as threats and challenges as well as opportunities that shape growth in Global CRISPR Technology Market.

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Few Points From TOC:1 Scope of the Report2 Executive Summary3 Global CRISPR Technology by Players4 CRISPR Technology by RegionsContinued

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Global CRISPR Technology Market is said to have a potential scope for growth in the years by 2025- Thermo Fisher Scientific, Merck KGaA, GenScript,...

CRISPR takes first steps in humans

CRISPR-Cas9 is a revolutionary gene-editing technology that offers the potential to treat diseases such as cancer, but the effects of CRISPR in patients are currently unknown. Stadtmauer et al. report a phase 1 clinical trial to assess the safety and feasibility of CRISPR-Cas9 gene editing in three patients with advanced cancer (see the Perspective by Hamilton and Doudna). They removed immune cells called T lymphocytes from patients and used CRISPR-Cas9 to disrupt three genes (TRAC, TRBC, and PDCD1) with the goal of improving antitumor immunity. A cancer-targeting transgene, NY-ESO-1, was also introduced to recognize tumors. The engineered cells were administered to patients and were well tolerated, with durable engraftment observed for the study duration. These encouraging observations pave the way for future trials to study CRISPR-engineered cancer immunotherapies.

Science, this issue p. eaba7365; see also p. 976

Most cancers are recognized and attacked by the immune system but can progress owing to tumor-mediated immunosuppression and immune evasion mechanisms. The infusion of ex vivo engineered T cells, termed adoptive T cell therapy, can increase the natural antitumor immune response of the patient. Gene therapy to redirect immune specificity combined with genome editing has the potential to improve the efficacy and increase the safety of engineered T cells. CRISPR coupled with CRISPR-associated protein 9 (Cas9) endonuclease is a powerful gene-editing technology that potentially allows the ability to target multiple genes in T cells to improve cancer immunotherapy.

Our first-in-human, phase 1 clinical trial (clinicaltrials.gov; trial NCT03399448) was designed to test the safety and feasibility of multiplex CRISPR-Cas9 gene editing of T cells from patients with advanced, refractory cancer. A limitation of adoptively transferred T cell efficacy has been the induction of T cell dysfunction or exhaustion. We hypothesized that removing the endogenous T cell receptor (TCR) and the immune checkpoint molecule programmed cell death protein 1 (PD-1) would improve the function and persistence of engineered T cells. In addition, the removal of PD-1 has the potential to improve safety and reduce toxicity that can be caused by autoimmunity. A synthetic, cancer-specific TCR transgene (NY-ESO-1) was also introduced to recognize tumor cells. In vivo tracking and persistence of the engineered T cells were monitored to determine if the cells could persist after CRISPR-Cas9 modifications.

Four cell products were manufactured at clinical scale, and three patients (two with advanced refractory myeloma and one with metastatic sarcoma) were infused. The editing efficiency was consistent in all four products and varied as a function of the single guide RNA (sgRNA), with highest efficiency observed for the TCR chain gene (TRAC) and lowest efficiency for the TCR chain gene (TRBC). The mutations induced by CRISPR-Cas9 were highly specific for the targeted loci; however, rare off-target edits were observed. Single-cell RNA sequencing of the infused CRISPR-engineered T cells revealed that ~30% of cells had no detectable mutations, whereas ~40% had a single mutation and ~20 and ~10% of the engineered T cells were double mutated and triple mutated, respectively, at the target sequences. The edited T cells engrafted in all three patients at stable levels for at least 9 months. The persistence of the T cells expressing the engineered TCR was much more durable than in three previous clinical trials during which T cells were infused that retained expression of the endogenous TCR and endogenous PD-1. There were no clinical toxicities associated with the engineered T cells. Chromosomal translocations were observed in vitro during cell manufacturing, and these decreased over time after infusion into patients. Biopsies of bone marrow and tumor showed trafficking of T cells to the sites of tumor in all three patients. Although tumor biopsies revealed residual tumor, in both patients with myeloma, there was a reduction in the target antigens NY-ESO-1 and/or LAGE-1. This result is consistent with an on-target effect of the engineered T cells, resulting in tumor evasion.

Preliminary results from this pilot trial demonstrate that multiplex human genome engineering is safe and feasible using CRISPR-Cas9. The extended persistence of the engineered T cells indicates that preexisting immune responses to Cas9 do not appear to present a barrier to the implementation of this promising technology.

T cells (center) were isolated from the blood of a patient with cancer. CRISPR-Cas9 ribonuclear protein complexes loaded with three sgRNAs were electroporated into the normal T cells, resulting in gene editing of the TRAC, TRBC1, TRBC2, and PDCD1 (encoding PD-1) loci. The cells were then transduced with a lentiviral vector to express a TCR specific for the cancer-testis antigens NY-ESO-1 and LAGE-1 (right). The engineered T cells were then returned to the patient by intravenous infusion, and patients were monitored to determine safety and feasibility. PAM, protospacer adjacent motif.

CRISPR-Cas9 gene editing provides a powerful tool to enhance the natural ability of human T cells to fight cancer. We report a first-in-human phase 1 clinical trial to test the safety and feasibility of multiplex CRISPR-Cas9 editing to engineer T cells in three patients with refractory cancer. Two genes encoding the endogenous T cell receptor (TCR) chains, TCR (TRAC) and TCR (TRBC), were deleted in T cells to reduce TCR mispairing and to enhance the expression of a synthetic, cancer-specific TCR transgene (NY-ESO-1). Removal of a third gene encoding programmed cell death protein 1 (PD-1; PDCD1), was performed to improve antitumor immunity. Adoptive transfer of engineered T cells into patients resulted in durable engraftment with edits at all three genomic loci. Although chromosomal translocations were detected, the frequency decreased over time. Modified T cells persisted for up to 9 months, suggesting that immunogenicity is minimal under these conditions and demonstrating the feasibility of CRISPR gene editing for cancer immunotherapy.

Gene editing offers the potential to correct DNA mutations and may offer promise to treat or eliminate countless human genetic diseases. The goal of gene editing is to change the DNA of cells with singlebase pair precision. The principle was first demonstrated in mammalian cells when it was shown that expression of a rare cutting endonuclease to create double-strand DNA breaks resulted in repair by homologous and nonhomologous recombination (1). A variety of engineered nucleases were then developed to increase efficiency and enable potential therapeutic applications, including zinc finger nucleases, homing endonucleases, transcription activatorlike effector nucleases, and CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats associated with Cas9 endonuclease) (2). The first pilot human trials using genome editing were conducted in patients with HIV/AIDS and targeted the white blood cell protein CCR5, with the goal of mutating the CCR5 gene by nonhomologous recombination and thereby inducing resistance to HIV infection (3, 4). The incorporation of multiple guide sequences in CRISPR-Cas9 permits, in principle, multiplex genome engineering at several sites within a mammalian genome (59). The ability of CRISPR to facilitate efficient multiplex genome editing has greatly expanded the scope of possible targeted genetic manipulations, enabling new possibilities such as simultaneous deletion or insertion of multiple DNA sequences in a single round of mutagenesis. The prospect of using CRISPR engineering to treat a host of diseases, such as inherited blood disorders and blindness, is moving closer to reality.

Recent advances in CRISPR-Cas9 technology have also permitted efficient DNA modifications in human T cells, which holds great promise for enhancing the efficacy of cancer therapy. T lymphocytes are specialized immune cells that are largely at the core of the modern-day cancer immunotherapy revolution. The T cell receptor (TCR) complex is located on the surface of T cells and is central for initiating successful antitumor responses by recognizing foreign antigens and peptides bound to major histocompatibility complex molecules. One of the most promising areas of cancer immunotherapy involves adoptive cell therapy, whereby the patients own T cells are genetically engineered to express a synthetic (transgenic) TCR that can specifically detect and kill tumor cells. Recent studies have shown safety and promising efficacy of such adoptive T cell transfer approaches using transgenic TCRs specific for the immunogenic NY-ESO-1 tumor antigen in patients with myeloma, melanoma, and sarcoma (1012). One limitation of this approach is that the transgenic TCR has been shown to mispair and/or compete for expression with the and chains of the endogenous TCR (1315). Mispairing of the therapeutic TCR and chains with endogenous and chains reduces therapeutic TCR cell surface expression and potentially generates self-reactive TCRs.

A further shortcoming of adoptively transferred T cells has been the induction of T cell dysfunction or exhaustion leading to reduced efficacy (16). Programmed cell death protein 1 (PD-1)deficient allogeneic mouse T cells with transgenic TCRs showed enhanced responses to alloantigens, indicating that the PD-1 protein on T cells plays a negative regulatory role in antigen responses that are likely to be cell intrinsic (17). The adoptive transfer of PD-1deficient T cells in mice with chronic lymphocytic choriomeningitis virus infection initially leads to enhanced cytotoxicity and later to enhanced accumulation of terminally differentiated T cells (18). Antibody blockade of PD-1, or disruption or knockdown of the gene encoding PD-1 (i.e., PDCD1), improved chimeric antigen receptor (CAR) or TCR T cellmediated killing of tumor cells in vitro and enhanced clearance of PD-1 ligandpositive (PD-L1+) tumor xenografts in vivo (1923). In preclinical studies, we and others found that CRISPR-Cas9mediated disruption of PDCD1 in human T cells transduced with a CAR increased antitumor efficacy in tumor xenografts (2426). Adoptive transfer of transgenic TCR T cells specific for the cancer antigen NY-ESO-1, in combination with a monoclonal antibody targeting PD-1, enhanced antitumor efficacy in mice (27). We therefore designed a first-in-human, phase 1 human clinical trial to test the safety and feasibility of multiplex CRISPR-Cas9 genome editing for a synthetic biology cancer immunotherapy application. We chose to target endogenous TRAC, TRBC, and PDCD1 on T cells to increase the safety and efficacy profile of NY-ESO-1 TCRexpressing engineered cells. In principle, this strategy allowed us to increase exogenous TCR expression and reduce the potential for mixed heterodimer formation (i.e., by deleting the and TCR domain genes TRAC and TRBC, respectively) and to limit the development of T cell exhaustion, which can be triggered by the checkpoint ligands PD-L1 and PD-L2 (i.e., by deleting PDCD1).

The phase 1 human trial (clinicaltrials.gov; trial NCT03399448) was designed to assess the safety and feasibility of infusing autologous NY-ESO-1 TCRengineered T cells in patients after CRISPR-Cas9 editing of the TRAC, TRBC, and PDCD1 loci. During the manufacturing process, cells were taken out of the cancer patient, engineered, and then infused back into the individual. The genetically engineered T cell product was termed NYCE (NY-ESO-1transduced CRISPR 3X edited cells) and is referred to as NYCE hereafter. During clinical development of the protocol, we elected to use a TCR rather than a CAR because the incidence of cytokine release syndrome is generally less prevalent using TCRs (11). In principle, this allowed a more discriminating assessment of whether gene editing with Cas9 was potentially immunogenic or toxic when compared with the baseline low level of adverse events observed in our previous clinical trial targeting NY-ESO-1 with transgenic TCRs (11). The autologous T cells were engineered by lentiviral transduction to express an HLA-A2*0201restricted TCR specific for the SLLMWITQC peptide in NY-ESO-1 and LAGE-1. The manufacturing process, vector design, and clinical protocol for NYCE T cells are described in the materials and methods and are depicted schematically (figs. S1 and S2). Of the six patients who were initially enrolled, four patients had successfully engineered T cells that were subjected to detailed release criteria testing as specified in the U.S. Food and Drug Administration (FDA)accepted Investigational New Drug application (table S1) (see fig. S3 for the consort diagram). Of the four patients with cell products available, one patient assigned unique patient number (UPN) 27 experienced rapid clinical progression and was no longer eligible for infusion owing to the inability to meet protocol-mandated safety criteria (see supplementary materials). Of the three patients who were infused with CRISPR-Cas9engineered T cells, two patients had refractory advanced myeloma and one patient had a refractory metastatic sarcoma not responding to multiple prior therapies (Table 1). The patients were given lymphodepleting chemotherapy with cyclophosphamide and fludarabine on days 5 to 3 (i.e., before administration with CRISPR-Cas9engineered T cells) and a single infusion of 1 108 manufactured CRISPR-Cas9engineered T cells per kilogram on day 0 of the protocol (fig. S2). No cytokines were administered to the patients.

MM, multiple myeloma; BM, bone marrow; XRT, radiation therapy; ASCT, autologous hematopoietic stem cell transplant; ND, not done.

The T cell product was manufactured by electroporation of ribonucleoprotein complexes (RNPs) comprising recombinant Cas9 loaded with equimolar mixtures of single guide RNA (sgRNA) for TRAC, TRBC, and PDCD1 followed by lentiviral transduction of the transgenic TCR (Fig. 1A). All products were expanded to >1 1010 T cells by the time of harvest (Fig. 1B). The transgenic TCR could be detected by flow cytometric staining for V8.1 or dextramer staining, ranging from 2 to 7% of T cells in the final product (Fig. 1C). The frequency of editing, as determined by digital polymerase chain reaction (PCR), varied according to the sgRNA and was about 45% for TRAC, 15% for TRBC, and 20% for PDCD1 (Fig. 1D). Final product transduction efficiency, CD4:CD8 ratio, and dosing are shown in table S2.

(A) Schematic representation of CRISPR-Cas9 NYCE T cells. (B) Large-scale expansion of NYCE T cells. Autologous T cells were transfected with Cas9 protein complexed with sgRNAs (RNP complex) against TRAC, TRBC (i.e., endogenous TCR deletion), and PDCD1 (i.e., PD-1 deletion) and subsequently transduced with a lentiviral vector to express a transgenic NY-ESO-1 cancer-specific TCR. Cells were expanded in dynamic culture for 8 to 12 days. On the final day of culture, NYCE T cells were harvested and cryopreserved in infusible medium. The total number of enriched T cells during culture is plotted for all four subjects (UPN07, UPN27, UPN35, and UPN39). (C) NY-ESO-1 TCR transduction efficiency was determined in harvested infusion products by flow cytometry. Data are gated on live CD3-expressing and V8.1- or dextramer-positive lymphocytes and further gated on CD4-positive and/or CD8-positive cells. (D) The frequencies of TRAC, TRBC, and PDCD1 gene-disrupted total cells in NYCE infusion products were measured using chip-based digital PCR. All data are representative of at least two independent experiments. Error bars represent mean SEM.

The potency of the final engineered T cells was assessed by coculture with HLA-A2+ tumor cells engineered to express NY-ESO-1 (Fig. 2A). The engineered T cells had potent antigen-specific cytotoxicity over a wide range of effector-totarget cell ratios. Interestingly, the cells treated with CRISPR-Cas9 were more cytotoxic than control cells transduced with the TCR but electroporated without CRISPR-Cas9 (i.e., cells that retained endogenous TCR). This is consistent with previous findings in mouse T cells, when a transgenic TCR was inserted into the endogenous locus, ablating expression of the endogenous TCR (15). Further studies will be required to determine if PD-1 knockout contributes to the increased potency afforded by knockout of the endogenous TCR.

(A) Cytotoxicity of NYCE T cells cocultured with HLA-A*0201positive Nalm-6 tumor cells engineered to express NY-ESO-1 and luciferase. Patient T cells transduced with the NY-ESO-1 TCR without CRISPR-Cas9 editing (NY-ESO-1 TCR) and untransduced T cells with CRISPR-Cas9 editing of TRAC, TRBC, and PDCD1 (labeled CRISPR) were included as controls (n = 4 patient T cell infusion products). Asterisks indicate statistical significance determined by paired Students t tests between groups (*P < 0.05). Error bars represent SEM. (B) Levels of soluble interferon- produced by patient NYCE T cell infusion products (labeled NYCE) after a 24-hour coculture with anti-CD3 and anti-CD28 antibody-coated beads or NY-ESO-1expressing Nalm-6 target cells. Patient NY-ESO-1 TCRtransduced T cells (NY-ESO-1 TCR) and untransduced, CRISPR-Cas9edited T cells (labeled CRISPR) served as controls. Error bars represent SEM. (C) Quantification of residual Cas9 protein in NYCE T cell infusion products in clinical-scale manufacturing is shown over time. Asterisks indicate statistical significance determined by paired Students t tests between time points (*P < 0.05). (D) Results from the fluorescence-based indirect ELISA screen performed to detect antibodies against Cas9 protein in the sera of three patients treated with NYCE T cells. Each dot represents the amount of anti-Cas9 signals detected in patient serum before T cell infusion (indicated by a vertical black arrow) and at various time points after NYCE T cell transfer. RFU, relative fluorescent units. (E) Immunoreactive Cas9-specific T cells in baseline patient leukapheresis samples were detected. Representative flow cytometry plots (left) from two patients whose T cells were positive for interferon- in response to Cas9 peptide stimulation. Unstimulated T cells treated with vehicle alone (dimethyl sulfoxide, DMSO) served as a negative control, whereas matched T cells stimulated with phorbol myristate acetate (PMA) and ionomycin served as a positive control. Bar graphs (right) show the frequency of ex vivo CD4+ and CD8+ T cells from patients or healthy donor controls (n = 6) that secrete interferon- in response to stimulation with three different Cas9 peptide pools. The background frequency of interferon-expressing T cells (unstimulated control group, DMSO alone) is subtracted from the values shown in the bar graph. Error bars represent SD.

We developed a sensitive immunoassay for detection of Streptococcus pyogenes Cas9 protein and quantified Cas9 early in the manufacturing process, showing declining levels that were <0.75 fg per cell in the harvested final product (Fig. 2C). Using a competitive fluorescence enzyme-linked immunosorbent assay (ELISA) screen, we found that healthy donors have humoral reactivity to Cas9 in serum (data not shown) and T cells (Fig. 2E), confirming previous reports (2830). Interestingly, we found that the three patients tested at a variety of time points after infusion of the engineered T cells did not develop humoral responses to Cas9. The lack of immunization to Cas9 is consistent with the extended persistence of the infused cells (Fig. 3) and could be a consequence of the low content of Cas9 in the infused product and/or to the immunodeficiency in the patients as a result of their extensive previous treatment histories (Table 1).

(A) The total number of vector copies per microgram of genomic DNA of the NY-ESO-1 TCR transgene in the peripheral blood (UPN07, UPN35, and UPN39), bone marrow (UPN07 and UPN35; multiple myeloma), and tumor (UPN39; sarcoma) is shown pre and postNYCE T cell infusion. (B) Calculated absolute numbers of NY-ESO-1 TCRexpressing T cells per microliter of whole blood from the time of infusion to various postinfusion time points in the study are shown. The limit of detection is about 2.5 cells per microliter of whole blood. (C) Frequencies of CRISPR-Cas9edited T cells (TRAC, TRBC, and PDCD1 knockout) before and after adoptive cell transfer are depicted. Error bars represent SD.

Three patients with advanced, refractory cancer were given infusions of the CRISPR-Cas9engineered T cells. The infusions were well tolerated, with no serious adverse events (Table 2); importantly, there were no cases of cytokine release syndrome, which is a potentially life-threatening systemic inflammatory response that has been associated with cancer immunotherapies (31). All three patients were infused with 1 108 cells/kg, and, owing to the considerable variation in TCR transduction efficiencies (table S2), the absolute number of infused engineered T cells ranged from 6.0 107 to 7.1 108 cells. Despite the variation in engineered cells, there were high peak levels and sustained persistence of the engineered cells in the blood of all three patients (Fig. 3A). The peak and steady-state levels of engineered cells were lowest in patient UPN35, who also had the lowest transduction efficiency (table S2). The persistence of the transduced cells is notably stable from 3 to 9 months after infusion, varying from 5 to 50 cells per microliter of blood (Fig. 3B). Using a subject-specific piecewise linear model, the decay half-lives of the transduced cells were 20.3, 121.8, and 293.5 days for UPN07, UPN35, and UPN39, respectively. The average decay half-life was 83.9 days (15 to 153 days, 95% confidence interval) for the three subjects, as estimated by a piecewise linear mixed-effects model that assumes cells decay linearly from day 14 postexpansion and random effects to allow varying level of expansion (or peak values) across subjects. The stable engraftment of our engineered T cells is notably different from previously reported trials with NY-ESO-1 TCRengineered T cells, in which the half-life of the cells in blood was ~1 week (11, 32, 33). Biopsy specimens of bone marrow in the myeloma patients and tumor in the sarcoma patient demonstrated trafficking of the engineered T cells to the tumor in all three patients at levels approaching those in the blood compartment (Fig. 3A).

indicates no adverse event.

To determine the engraftment frequency of the CRISPR-Cas9 gene-edited cells, we initially used chip-based digital PCR. With this assay, engraftment of cells with editing at the TRAC and PDCD1 loci was evident in all three patients (Fig. 3C). There was sustained persistence of TRAC and PDCD1 edits in patients UPN39 and UPN07 at frequencies of 5 to 10% of circulating peripheral blood mononuclear cells (PBMCs), whereas TRBC-edited cells were lowest in frequency and only transiently detected. The low-level engraftment of TRBC-edited cells is likely related to the observation that this locus had the lowest level of editing efficiency in our preclinical studies (25) and in the harvested products (Fig. 1D).

On- and off-target editing efficiency was assessed in the NYCE cells at the end of product manufacturing. Details of the analysis for UPN07 are shown as an example in Fig. 4, with detailed analysis of the other three manufactured products shown in table S3. The average on-target CRISPR-Cas9 editing efficiency for all engineered T cell products for each target is shown in Table 3. We used iGUIDE (34), a modification of the GUIDE sequencing (GUIDE-seq) method (35), to analyze the Cas9-mediated cleavage specificity. A complication of assays to assess repair by nonhomologous end joining (NHEJ) is that DNA double-strand breaks are formed spontaneously during cell division at high rates in the absence of added nucleases (36), which can increase the background in assays of off-target cleavage. The distribution of on- and off-target cleavage is expected to vary for the three sgRNAs that were used in the manufacturing process (fig. S1A). Of the three sgRNAs, there were more off-target mutations identified for TRBC than for the other loci (Fig. 4C and figs. S4 and S5). The sgRNA for PDCD1 was the most specific, because very few off-target edits were identified in more than 7000 sites of cleavage and there were very few off-target reads identified at the TRAC1 and TRAC2 loci (Fig. 4C).

(A) Genomic distribution of oligonucleotide (dsODN) incorporation sites, which mark locations of double-strand breaks. The ring indicates the human chromosomes aligned end to end, plus the mitochondrial chromosome (labeled M). The targeted cleavage sites are on chromosomes 2, 7, and 14. The frequency of cleavage and subsequent dsODN incorporation is shown on a log scale on each ring (pooled over 10-Mb windows). The purple innermost ring plots all alignments identified. The green ring shows pileups of three or more overlapping sequences, the blue ring shows alignments extending along either strand from a common dsODN incorporation site (flanking pairs), and the red ring shows reads with matches to the gRNA (allowing <6 mismatches) within 100 bp (target matched). (B) Distribution of inferred positions of cleavage and dsODN incorporation at an on-target locus. Incorporations in different strand orientations are shown on the positive (red) and negative (blue) y axis. The percentage in the bottom right corner is an estimate of the number of incorporations associated with the on-target site (based on pileups) captured within the allowed window of 100 bp. (C) Sequences of sites of cleavage and dsODN incorporation are shown, annotated by whether they are on target or off target (Target); the total number of unique alignments associated with the site (Abund.); and an identifier indicating the nearest gene (Gene ID). An asterisk after the gene name indicates that the site is within the transcription unit of the specific gene, whereas ~ indicates that the gene appears on the allOnco cancer-associated gene list.

The genomic localization of identified DNA cleavage sites was as expected, given the chromosomal location of the three targeted genes on chromosomes 2, 7, and 14 (Fig. 4A). The distribution of the incorporation of the double-stranded oligodeoxynucleotide (dsODN) label around on-target sites, based on pileups within a window of 100 base pairs (bp), is shown in Fig. 4B and fig. S4. Although most mutations were on target, there were off-target mutations identified (Fig. 4C and fig. S5). For the TRAC sgRNA, there were low-abundance mutations within the transcriptional unit of CLIC2 (chloride intracellular channel 2); however, disruption of CLIC2 in T cells is not expected to have negative consequences because it is not reported to be expressed in T cells. For the TRBC sgRNA, off-target edits were identified in genes encoding a transcriptional regulator (ZNF609) and a long intergenic nonprotein coding RNA (LINC00377) (table S3). In addition to the above post hoc investigations of multiplex editing specificity, all products were shown not to have cellular transformation by virtue of the absence of long-term growth before infusion (table S1).

In addition to the above detection of repair of double-strand DNA breaks by NHEJ, on-target mutagenesis by engineered nucleases can result in deletions, duplications, inversions, and translocations and can also lead to complex chromosomal rearrangements under some conditions (37). CRISPR-Cas9 has been used to intentionally create oncogenic chromosomal rearrangements (38). In preclinical studies with human T cells, simultaneous gene editing of TRAC and CD52 using TALENs led to translocations that were detected at frequencies of 104 to 102 (39). In a subsequent clinical report using dual-gene editing with TALENs, chromosomal rearrangements were observed in 4% of infused cells (40). To study the safety and genotoxicity of multiplex CRISPR-Cas9 genome editing on three chromosomes, we used stringent release criteria of the manufactured cells and assays to detect translocations (fig. S6). We developed and qualified quantitative PCR (qPCR) assays to quantify the 12 potential translocations that could occur with the simultaneous editing of four loci: TRAC, TRBC1, TRBC2, and PDCD1 (see materials and methods). We observed translocations in all manufactured products; however, the translocations were at the limit of detection for the assay in patient UPN39 (Fig. 5A). TRBC1:TRBC2 was the most abundant rearrangement (Fig. 5A), resulting in a 9.3-kb deletion (supplementary materials). The deletion and translocations peaked on days 5 to 7 of manufacturing and then declined in frequency until cell harvest. The translocations and the TRBC1:TRBC2 deletion were evident in the three patients between 10 days after infusion and 30 to 170 days after infusion (Fig. 5B). However, the rearrangements declined in frequency in vivo, suggesting that they conferred no evidence of a growth advantage over many generations of expansion in the patients on this trial (Fig. 3, A and B). At days 30, 150, and 170 in patients UPN07, UPN35, and UPN39, respectively, chromosomal translocations were at the limits of detection or not detected for all rearrangements except for the 9.3-kb deletion for TRBC1:TRBC2.

(A) Evaluation of chromosomal translocations in NYCE T cell infusion products during the course of large-scale culture is shown. For the 12 monocentromeric translocation assays conducted, a positive reference sample that contains 1 103 copies of the synthetic template plasmid was evaluated as a control, and the percent difference between expected and observed marking was calculated. The absence of amplification from the 12 reactions that correspond to the different chromosomal translocations indicates assay specificity (see methods). (B) Longitudinal analysis of chromosomal translocations in vivo in three patients pre and postNYCE T cell product infusion is displayed. In (A) and (B), error bars represent SD. For graphical purposes, the proportions of affected cells were plotted on a log scale; a value of 0.001% indicates that translocations were not detected.

We used single-cell RNA sequencing (scRNA-seq) to comprehensively characterize the transcriptomic phenotype of the NYCE T cells and their evolution over time in patient UPN39 (fig. S7). UPN39 was chosen because they had the highest level of cell engraftment and because this patient had evidence of tumor regression. CRISPR-Cas9engineered T cells were infused to patient UPN39 and recovered after infusion from the blood on day 10 and at ~4 months (day 113) and were analyzed by scRNA-seq, as described in the materials and methods. For each sample (infusion product, day 10 and day 113), T cells were sorted on the basis of expression of CD4 or CD8 and processed using droplet-based 5 scRNA-seq. From the gene expression libraries, PCR was used to further amplify cellular cDNA corresponding to the NY-ESO-1 TCR transgene, as well as TRAC, TRBC, and PDCD1 target sequences, allowing us to genotype single cells as wild type or mutant. In the infusion product, cells were identified that contained mutations in all three target sequences (Fig. 6, A and B). The most commonly mutated gene was TRAC. About 30% of cells had no mutations identified, whereas ~40% had one mutation, and ~20 and ~10% of the T cells in the manufactured product were double mutated and triple mutated, respectively, at the target sequences. Of the transgenic TCR+ cells in the infusion product, monogenic mutations were less frequent than digenic and trigenic mutations (Fig. 6A). Single-cell genotyping of UPN39 cells at 10 days and 4 months after infusion showed a decline in the frequency of gene-edited T cells from the levels in the infusion product, and this decline occurred regardless of whether the cells were transduced with the NY-ESO-1 TCR (Fig. 6C). The frequency of gene-edited cells was quite stable between day 10 and 4 months postinfusion, and notably, about 40% of the peripheral bloodcirculating T cells in this patient 4 months after infusion were mutated at any one of the targeted genes (Fig. 6, B and C, and table S4).

(A) Venn diagram showing relative numbers of NY-ESO-1 TCRpositive cells with TRAC, TRBC, and/or PDCD1 mutations in the NYCE T cell infusion product (IP) (day 0). (B) Proportions of preinfusion (IP, day 0) and postinfusion (days 10 and 113) wild-type T cells with TRAC, TRBC, or PDCD1 mutations or expressing the NY-ESO-1 TCR transgene. Numbers of cells belonging to each of these categories are listed below the graph. (C) Analysis of NY-ESO-1 TCRpositive (right) and NY-ESO-1 TCRnegative (left) cells without mutations (wild type) or with single, double, or triple mutations at day 0 (NYCE T cell infusion product) and day 113 postNYCE T cell infusion. Numbers of analyzed cells for each time point are listed above the bars. (D) Uniform manifold approximation and projection (UMAP) plots of gene expression data. Analysis was performed on all T cells integrated across time points, but only NY-ESO-1 TCRexpressing cells, split by time point, are shown (top). The increase in TCF7 expression is indicative of an acquired central memory phenotype (bottom, same cells). (E) Heatmap showing scaled expression of differentially expressed genes in NY-ESO-1 TCRpositive T cells across time points. Color scheme is based on scaled gene expression from 2 (purple) to 2 (yellow).

Of particular interest is the frequency and evolution of PD-1deficient T cells owing to the previous mention that genetic disruption of PDCD1 in CAR and TCR T cells enhances antitumor efficacy in preclinical models (19, 2124). We found that ~25% of the T cells expressing the NY-ESO-1 TCR in the infusion product had mutations in the PDCD1 locus (fig. S8). It is interesting that the frequency of cells with edits in the PDCD1 locus decreased to ~5% of the cells expressing the transgenic TCR at 4 months postinfusion. This would be consistent with mouse studies of chronic infection in which PD-1deficient T cells are less able to establish memory (18).

Figure 6D shows the distribution of engineered T cells expressing the NY-ESO-1 TCR transgene in the infusion product of patient UPN39, and again at 4 months in vivo as they evolve from the infused cells. In the heatmap (Fig. 6E), the most differentially expressed genes in the cells expressing the NY-ESO-1 transcript at the various time points are shown in table S5. Notably, UPN39 had increases in expression of genes associated with central memory (IL7R and TCF7) over time (Fig. 6, D and E, and table S4). This is in marked contrast to the recently published results with NY-ESO-1 T cells in the absence of genome editing, in which the infused transgenic T cells evolved to a terminally differentiated phenotype and displayed characteristics of T cell exhaustion in cancer patients (12).

The clinical course of the three infused cancer patients is shown in Fig. 7 (and described in the materials and methods). No patient experienced cytokine release syndrome or overt side effects attributed to the cell infusion (table S5). The best clinical responses were stable disease in two patients. UPN39 had a mixed response, with a ~50% decrease in a large abdominal mass that was sustained for 4 months (Fig. 7D), although other lesions progressed. As of December 2019, all patients have progressed: Two are receiving other therapies, and UPN07 died from progressive myeloma.

(A) Swimmers plot describing time on study for each patient, duration of follow-up off study (defined as survival beyond progression or initiation of other cancer therapy), and present status (differentially colored) is shown. Arrows indicate ongoing survival. SD, stable disease; PD, progressive disease. (B) Changes in kappa light chain levels (mg/liter 103) in patient UPN07 after NYCE T cell product infusion are depicted. Vertical black arrow indicates initiation of a D-ACE salvage chemotherapy regimen (defined as intravenous infusion of cisplatin, etoposide, cytarabine, and dexamethasone). (C) Longitudinal M-spike levels (g/dl) in patient UPN35 postNYCE T cell product administration are shown. Vertical black arrows indicate administration of combination therapy with elotuzumab, pomalidomide, and dexamethasone. (D) Computed tomography scans demonstrating tumor regression in patient UPN39 after administration of an autologous NYCE T cell infusion product. Radiologic studies were obtained before therapy and after adoptive transfer of NYCE T cells. Tumor is indicated by red X. (E) Cytolytic capacity of NY-ESO-1specific CD8+ T cells recovered at the indicated month after infusion and expanded from patients is shown. PBMC samples collected after NYCE T cell product infusion were expanded in vitro in the presence of NY-ESO-1 peptide and interleukin-2. The ability of expanded effector cells to recognize antigen and elicit cytotoxicity was tested in a 4-hour 51Cr release assay incorporating Nalm-6 NY-ESO-1+, parental Nalm-6 (NY-ESO-1), and A375 melanoma cells (NY-ESO-1+). All target cell lines were HLA-A*02 positive. Assays were performed in triplicate, and error bars represent SD.

Biopsies of bone marrow and tumor showed trafficking of the NYCE-engineered T cells to the sites of tumor in all three patients (Fig. 3A). It is interesting to note that even though the tumor biopsies revealed residual tumor, in both patients with myeloma, there was a reduction in the target antigens NY-ESO-1 and/or LAGE-1 (fig. S9). The reduction of target antigen was transient in patient UPN07 and persistent in patient UPN35. This result is consistent with an on-target effect of the infused cells, likely resulting in tumor editing (41).

To determine whether the NYCE cells retained antitumor activity after infusion, samples of blood obtained from patients 3 to 9 months after infusion were expanded in culture in the presence of NY-ESO-1 peptide and assessed for cytotoxicity against tumor cells (Fig. 7E and fig. S10). Antigen-specific cytotoxicity was observed in all three patients. It is interesting to note that the most potent antitumor cytotoxicity was observed in UPN39, because UPN39 was the only patient to have tumor regression after infusion of the CRISPR-Cas9engineered T cells (Fig. 7D).

Our phase 1 first-in-human pilot study demonstrates the initial safety and feasibility of multiplex CRISPR-Cas9 T cell human genome engineering in patients with advanced, refractory cancer. In one patient analyzed at depth, a frequency of 30% of digenic and trigenic editing was achieved in the infused cell population, and 20% of the TCR transgenic T cells in circulation 4 months later had persisting digenic and trigenic edits. We chose to redirect specificity of the T cells with a T cell receptor, rather than a CAR, to avoid the CAR-associated potential toxicities such as cytokine release syndrome (31). This provided a lower baseline toxicity profile, thus enhancing the ability to detect toxicity specifically associated with the CRISPR-Cas9engineering process. We observed mild toxicity, and most of the adverse events were attributed to the lymphodepleting chemotherapy. We note that although the initial clinical results have acceptable safety, experience with more patients given infusions of CRISPR-engineered T cells with higher editing efficiencies, and longer observation after infusion, will be required to fully assess the safety of this approach.

Our large-scale product manufacturing process resulted in gene-editing efficiencies similar to those in our preclinical studies (24). A surprising finding was the high-level engraftment and long-term persistence of the infused CRISPR-Cas9engineered T cells. In previous clinical studies testing adoptively transferred NY-ESO-1 transgenic T cells, the engrafted cells had an initial decay half-life of about 1 week (1012). The explanation for the extended survival that we observed remains to be determined and could include the editing of the endogenous TCR, PD-1, and/or the choice of the TCR and vector design.

The use of scRNA-seq technology permitted the analysis of the transcriptome of the infused NY-ESO-1specific T cells (i.e., CRISPR-Cas9engineered T cells) at baseline and for up to 4 months in vivo. The results shown for UPN39 revealed that the infused cells evolved to a state consistent with central memory. These results are in contrast to a recent study in which the infused NY-ESO-1 T cells evolved to a state consistent with T cell exhaustion (12). A limitation of our in vivo single-cell analysis is that for purposes of feasibility, it is limited to the one patient who had the highest level of engraftment. Another limitation is that we were not able to compare the transcriptional state of the modified cells in the tumor microenvironment with circulating NYCE T cells.

Analysis of the manufacturing process in vitro demonstrated monochromosomal translocations and rearrangements, and some of these persisted in vivo. The translocations were not random in occurrence and occurred most frequently between PDCD1:TRAC and TRBC1:TRBC2. The frequency of translocations that we observed with trigenic editing is similar to that reported for digenic editing using TALEN-mediated gene editing in preclinical and clinical studies, in which rearrangements were detected in about 4% of cells (39, 40). It is important to note that healthy individuals often harbor oncogenic translocations in B and T cells (4244). T cells bearing translocations can persist for months to years without evidence of pathogenicity (4547).

Antagonism of the PD-1:PD-L1 costimulatory pathway can result in organ-specific and systemic autoimmunity (17, 48). PD-1 has been reported to function as a haploinsufficient tumor suppressor in mouse T cells (49). Our patients have had engraftment with PD-1deficient T cells, and to date, there is no evidence of autoimmunity or T cell genotoxicity.

In conclusion, our phase 1 human pilot study has confirmed that multiplex CRISPR-Cas9 editing of the human genome is possible at clinical scale. We note that although the initial clinical results suggest that this treatment is safe, experience with more patients given infusions with higher editing efficiencies and longer observation after infusion will be required to fully assess the safety of this approach. The potential rejection of infused cells due to preexisting immune responses to Cas9 (28, 29) does not appear to be a barrier to the application of this promising technology. Finally, it is important to note that our manufacturing was based on the reagents available in 2016, when our protocol had been reviewed by the National Institutes of Health (NIH) Recombinant DNA Advisory Committee and received approval. Our Investigational New Drug application was subsequently reviewed and accepted by the FDA. There has been rapid progress in the field since that time, with the development of reagents that should increase efficiencies and decrease off-target editing using CRISPR-based technology (50).

The clinical protocol is listed at clinicaltrials.gov, trial NCT03399448. Protocol no. 1604-1524 Phase 1 trial of autologous T cells engineered to express NY-ESO-1 TCR and CRISPR gene edited to eliminate endogenous TCR and PD-1 (NYCE T Cells) was reviewed and approved by the U.S. National Institutes of Health Recombinant DNA Advisory Committee on 21 June 2016. See fig. S1B for clinical trial design. Patient demographics are shown in Table 1. A list of adverse events is depicted in Table 2.

The genomic gRNA target sequences with protospacer adjacent motif (PAM) underlined were: TRAC1 and TRAC2: 5-TGTGCTAGACATGAGGTCTATGG-3, TRBC: 5-GGAGAATGACGAGTGGACCCAGG-3, and PDCD1: 5-GGCGCCCTGGCCAGTCGTCTGGG-3. In vitro transcribed gRNA was prepared from linearized DNA (Aldevron) using Bulk T7 Megascript 5X (Ambion) and purified using RNeasy Maxi Kit (Qiagen).

Cas9 recombinant protein derived from S. pyogenes was TrueCut Cas9 v2 (catalogue no. A36499, ThermoFisher). Cas9 RNP was made by incubating protein with gRNA at a molar ratio of 1:1 at 25C for 10 min immediately before electroporation.

The 8F TCR recognizes the HLA-A*0201 SLLMWITQC epitope on NY-ESO-1 and LAGE-1. The 8F TCR was isolated from a T cell clone obtained from patient after vaccination with NY-ESO-1 peptide. The TCR sequences were cloned into a transfer plasmid that contains the EF-1 promoter, a cPPT sequence, a Rev response element and a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), as shown in fig. S1B. Plasmid DNA was manufactured at Puresyn, Inc. Lentiviral vector was produced at the University of Pennsylvania Center for Advanced Retinal and Ocular Therapeutics using transient transfection with four plasmids expressing the transfer vector, Rev, VSV-G, and gag-pol, in human embryonic kidney 293T cells.

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CRISPR-engineered T cells in patients with refractory cancer - Science Magazine

CRISPR Therapeutics (NASDAQ:CRSP) has done very well for itself, with the company's stock almost doubling in 2019 as optimism surrounding its gene-editing technology continues to grow. Considering how much of a game-changer gene-editing technology can be for patients with incurable conditions, it makes sense that people are excited.

While shares have tumbled over the past couple of months, this might be a good thing for investors looking to buy this promising stock at a cheaper price. If you're on the fence about CRISPR or are looking for a stock with great upside potential, here are a few reasons why CRISPR looks like a good pick in 2020.

Image source: Getty Images.

CRISPR is arguably the top name in the relatively young gene-editing market right now. The company currently has nine drug candidates, with four either having begun clinical testing or close to starting.

CRISPR's flagship candidate is CTX001, a drug that targets sickle cell disease and transfusion-dependent beta-thalassemia (TBT). Patients with either of these conditions have malformed red blood cells that struggle to deliver oxygen throughout the body. Approximately 300,000 infants are born with sickle cell disease each year, with another 60,000 born annually with TBT.At present, there are no treatments for either condition.

The other three noteworthy candidates in CRISPR's pipeline are CTX110, CTX120, and CTX130. These drug candidates are a type of new cancer-immunology treatment known as a chimeric antigen receptor T cell (CAR-T) therapy. These types of treatments involve modifying a patient's immune cells in a lab to make them better at killing cancer cells. While it's traditionally quite expensive, CRISPR's technology could possibly make these new CAR-T therapies cheaper than the competition.

While the drug is still in early clinical testing, CRISPR reported some success with CTX001 back in November when it announced that two patients had been treated successfully with the drug. The two patients, one diagnosed with sickle cell disease and the other with TBT, managed to eliminate all of their symptoms following a single CTX001 infusion.

In the case of TBT, the number of required blood transfusions dropped to zero, while the sickle cell disease patient experienced zero occlusive crises (blood vessel blockages that occur due to the abnormal shape of the patient's blood vessels).

CRISPR has confirmed that it will be providing more data for both CTX001 and its cancer immunotherapies sometime this year. That means that investors can look forward to further potential catalysts in 2020, likely toward the latter half of the year.

In general, investors shouldn't take too much stock in the financial figures of early-stage biotechstocks unless there's something really alarming going on (like not having enough cash). Revenue figures change dramatically once a drug receives approval, and companies tend to report significant losses until drug candidates reach late clinical stages.

However, CRISPR's situation is different. In its recently released fourth-quarter financial results, the company reported an impressive $77 million in revenue, a substantial improvement from the mere $100,000 seen last year. Annual revenue for 2019 came in at $289.6 million in comparison to 2018's $3.1 million.

While this virtually all comes from CRISPR's collaboration agreement with Vertex Pharmaceuticals, the important point is that CRISPR is now reporting a profit. Net income for the fourth quarter came in at $30.5 million, whereas last year the company saw a net loss of $47.6 million.

Data source: YCharts, CRISPR Therapeutics.

No other notable gene-editing stock out there is reporting a profit right now. Even if CRISPR ends up dipping into a net loss again in subsequent quarters, the fact that the company managed to report a positive net income this early on in its drug development program is impressive.

Given how young the gene-editing industry is and how experimental this technology can be, positive clinical results in this field can have a positive effect on all stocks in the sector. When Intellia Therapeuticspresented new data regarding two of its drug-editing programs earlier in February, shares of all gene-editing stocks -- including CRISPR -- shot up, despite the fact that they are all competitors.

While this might seem strange at first, it makes sense given how young this industry is. Further clinical proof that gene-editing drugs work, no matter where it comes from, is good for the entire sector. A rising tide lifts all ships, and CRISPR investors also should look out for potential catalysts from other gene-editing companies, which could act as an indirect catalyst for CRISPR's stock.

Intellia, Editas Medicine, and Sangamo Therapeuticsare all working on sickle cell disease and transfusion-dependent beta-thalassemia treatments of their own. Positive developments from their treatments could have a spillover effect on CRISPR's stock. Editas stated recently that it expects to file an Investigational New Drug (IND) application for EDIT-301, its sickle cell drug, by the end of 2020.

The answer is yes, it definitely can. CRISPR Therapeutics has plenty of good things going for it, and there is a lot of long-term enthusiasm surrounding both the company and the industry. While shares of CRISPR have fallen a fair bit over the past couple of months -- down 14% since the start of 2020-- so have other gene-editing stocks. As such, it doesn't seem to be as much of a problem with CRISPR in particular as it is a sector-wide phenomenon. Since there's no real news that appears to be behind this decline, I wouldn't worry about it too much.

Instead, now looks like a good time to buy gene-editing stocks, because they're trading at a bit of a discount. Back in November, Oppenheimer analyst Silvan Turkcan issued a price target of $80 for CRISPR Therapeutics, suggesting at least a 57.1% upside to the stock based on current prices. That seems very reasonable, and I wouldn't be surprised if CRISPR does much better than that in 2020.

However, CRISPR still remains a high-risk investment given the fact it's an early-stage biotech stock. If you want to buy shares right now, keep your position on the smaller side. Never risk too much of your portfolio on a single stock, no matter how promising it might seem.

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Can CRISPR Therapeutics Double Your Money in 2020? - The Motley Fool

ZUG, Switzerland and CAMBRIDGE, Mass., Feb. 26, 2020 (GLOBE NEWSWIRE) -- CRISPR Therapeutics (CRSP), a biopharmaceutical company focused on creating transformative gene-based medicines for serious diseases, today announced it proposes to elect Doug Treco, Ph.D. to its Board of Directors at the Companys upcoming annual general meeting to be held later this year. The Company also announced that Pablo Cagnoni, M.D., Chief Executive Officer of Rubius Therapeutics, will resign from the Board of Directors to focus on other commitments, effective immediately.

On behalf of our Board of Directors and management team, I would like to thank Pablo for his years of service and his many contributions to CRISPR Therapeutics, and I wish him the best in his future endeavors, said Rodger Novak, M.D., President and Chairman of the Board of CRISPR Therapeutics. We are grateful for his thoughtful guidance and support over the years.

Dr. Novak added: We are excited to invite Doug to our Board during an important time in CRISPR Therapeutics continued evolution. He has an impressive track record of success in advancing the development of numerous drug candidates, with a unique focus on rare disease, gene targeting, and gene therapy. His deep expertise and leadership experience will make him an outstanding addition to our Board, and we look forward to the valuable insights he will bring.

Doug co-founded Ra Pharmaceuticals, Inc. (RARX) in 2008 and has been Chief Executive Officer and a member of the Board of Directors since its inception. Ra Pharma is a leader in macrocyclic peptide and small molecule therapeutics targeting the complement pathway and has advanced its lead molecule, zilucoplan, into the clinic for multiple neuromuscular indications, including an ongoing pivotal Phase 3 study in myasthenia gravis. In October 2019, Ra Pharma entered into a merger agreement with UCB pursuant to which UCB will acquire Ra Pharma. He was an Entrepreneur-in-Residence at Morgenthaler Ventures from January 2008 to May 2014. In 1988, Doug co-founded Transkaryotic Therapies Inc. (TKT), a multi-platform biopharmaceutical company developing protein and gene therapy products, where he led the discovery of a number of approved biopharmaceuticals, including Dynepo, Replagal, Elaprase, and Vpriv. TKT (formerly Nasdaq: TKTX) was acquired by Shire Pharmaceuticals Group plc in 2005. He was a Visiting Scientist in the Department of Molecular Biology at Massachusetts General Hospital and a Lecturer in Genetics at Harvard Medical School from 2004 to 2007. Doug received his Ph.D. in Biochemistry and Molecular Biology from the State University of New York at Stony Brook and performed postdoctoral studies at the Salk Institute for Biological Studies and Massachusetts General Hospital.

About CRISPR TherapeuticsCRISPR Therapeutics is a leading gene editing company focused on developing transformative gene-based medicines for serious diseases using its proprietary CRISPR/Cas9 platform. CRISPR/Cas9 is a revolutionary gene editing technology that allows for precise, directed changes to genomic DNA. CRISPR Therapeutics has established a portfolio of therapeutic programs across a broad range of disease areas including hemoglobinopathies, oncology, regenerative medicine and rare diseases. To accelerate and expand its efforts, CRISPR Therapeutics has established strategic partnerships with leading companies including Bayer, Vertex Pharmaceuticals and ViaCyte, Inc. CRISPR Therapeutics AG is headquartered in Zug, Switzerland, with its wholly-owned U.S. subsidiary, CRISPR Therapeutics, Inc., and R&D operations based in Cambridge, Massachusetts, and business offices in San Francisco, California and London, United Kingdom. For more information, please visit http://www.crisprtx.com.

Important Additional Information and Where to Find ItCRISPR Therapeuticswill file a proxy statement with theUnited States Securities and Exchange Commission(SEC) in connection with the solicitation of proxies for its 2020 annual general meeting (2020 Annual Meeting). SHAREHOLDERS ARE STRONGLY ADVISED TO READ THE PROXY STATEMENT WHEN IT BECOMES AVAILABLE BECAUSE IT WILL CONTAIN IMPORTANT INFORMATION. Shareholders may obtain a free copy of the proxy statement, any amendments or supplements to the proxy statement and other documents thatCRISPR Therapeutics files with theSECfrom the SECs website atwww.sec.govor CRISPR Therapeutics website atwww.crisprtx.comas soon as reasonably practicable after such materials are electronically filed with, or furnished to, theSEC.

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Certain Information Regarding ParticipantsCRISPR Therapeutics, its directors, nominees for election as director, executive officers and other persons related toCRISPR Therapeutics may be deemed to be participants in the solicitation of proxies from CRISPR Therapeutics shareholders in connection with the matters to be considered at the 2020 Annual Meeting. Information concerning the interests of CRISPR Therapeutics participants in the solicitation is set forth in the materials filed byCRISPR Therapeutics with theSEC, including in its definitive proxy statement filed with theSEConApril 30, 2019, and will be set forth in the proxy statement relating to the 2020 Annual Meeting when it becomes available.

Investor Contact:Susan Kimsusan.kim@crisprtx.com

Media Contact:Rachel EidesWCG on behalf of CRISPR617-337-4167 reides@wcgworld.com

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CRISPR Therapeutics Proposes Changes to the Board of Directors - Yahoo Finance

The Global CRISPR And CRISPR-Associated (Cas) Genes Market report by Globalmarketers.Biz sets out the production, consumption, revenue, gross margin, cost, gross, market share, CAGR, and global market influencing factors of the market for 2020-2025. The segmentation of regional market included the historical and forecast mandates for North America, Europe, Asia-Pacific, Latin America, the Middle East and Africa. The CRISPR And CRISPR-Associated (Cas) Genes Market report provides a far-reaching industry analysis by types, applications, players and regions.

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The Top Key Players Are Covered In This Report Are As Follows:

Caribou BiosciencesAddgeneCRISPR THERAPEUTICSMerck KGaAMirus Bio LLCEditas MedicineTakara Bio USAThermo Fisher ScientificHorizon Discovery GroupIntellia TherapeuticsGE Healthcare Dharmacon

Above are the leading companies and brands that are driving the CRISPR And CRISPR-Associated (Cas) Genes Market. The CAGR numbers are looking quite impressive for the forecast period of 2020-2025 in the CRISPR And CRISPR-Associated (Cas) Genes Market. The sales, import, export and revenue figures are also skyrocketing in the forecast period. The key players and brands are making their moves by product launches, their researches, their joint ventures, merges, and accusations and are getting successful results. Complete study compiled with over 100+ pages, list of tables & figures, profiling 10+ companies.

Market Segment by Type, covers

Genome EditingGenetic engineeringgRNA Database/Gene LibrarCRISPR PlasmidHuman Stem CellsGenetically Modified Organisms/CropsCell Line Engineering

Market Segment by Applications, can be divided into

Biotechnology CompaniesPharmaceutical CompaniesAcademic InstitutesResearch and Development Institutes

Market Segment by Regions, regional analysis covers

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CRISPR And CRISPR-Associated (Cas) Genes Market Report Structure at a Glance:

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Table of Content:

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Global CRISPR And CRISPR-Associated (Cas) Genes Market Is Set to Boom in 2020,Coming Years - News Times

Agriculture is responsible for the production of a quarter of the total human-generated greenhouse gases. Growing food also uses about 70 percent of the water available to us. Moreover, agriculture (especially meat production) is the single most significant driver of deforestation and biodiversity loss. Food production is detrimental to the health of the planetbut it doesnt end there. Once the food reaches plates, poor-quality diets cause malnutrition, claiming more lives than tobacco, drug and alcohol combined.

Search for malnutrition online and you will see pictures of frail and sick children. But along with stunting, wasting, vitamin and mineral deficiency, malnutrition also includes overweight, obesity and other diet-related illnesses. Yes, 1 in 9 people around the world go to sleep hungry, but nearly 2 billion adults are also overweight or obese. As such, more than one-third of the world population suffers from at least one form of malnutrition.

With the climate and biodiversity crises, and the global public-health crisis in the form of malnutrition, we must find a healthy and environmentally sustainable diet to feed the growing population. In 2019, the EAT-Lancet Commission brought together leading experts in nutrition, health, sustainability and policy to recommend ways to transform the global food system to achieve a healthy and sustainable diet.

The EAT-Lancet report recommends that planetary health diets to feed 10 billion people by 2050 requires cutting down meat consumption by half and eating twice as much as fruits, vegetables, beans and nuts. Despite recognizing the need to make healthy food affordable for the poor, the EAT-Lancet Commission didnt review the cost and affordability of the ideal diet. Therefore, in a recent global study, scientists reviewed prices for nearly 750 food items to calculate the value of healthy and sustainable diets in 159 countries.

The research, published in Lancet Global Health, shows that many people in low and lower-middle-income countries are too poor to afford EAT-Lancets ideal diet. EAT-Lancet says that we would need to eat twice as much as many fruits and vegetables, and get more protein and fats from plant-source foods. However, the new study found that fruits, vegetables, beans and nuts are the most expensive items of the ideal diet accounting for half of its total price.

Shifting foodsystems

A key challenge of the 21st-century is to change our food system to produce a healthy diet that is both economically and environmentally sustainable. As EAT-Lancets ideal diet isnt affordable for much of the worlds low-income population, authorities must make several parallel interventions to tackle global food inequality.

Lower food prices and higher earnings would give poor people more purchasing power. We must also find cheaper, nutritious food alternatives that are affordable and accessible to people living in low-income areas. I believe that biotechnology has the power to lower the cost of locally and globally grown food, making the ideal diet economically viable to those that need it the most.

One problem is the lack of available, affordable options, which partly stems from decreasing agrobiodiversity. Just three crops (rice, wheat and corn) provide over half of the plant-derived calories worldwide. Shifting calories away from the starchy staple foods towards more nutritious fruits, vegetables and other protein-sourced food remains a significant challenge in meeting EAT-Lancet targets. Grand challenges require great technological solutions, and genetic engineering technology is among the most powerful tools at our disposal.

Power of biotechnology

Biotechnology can improve agrobiodiversity and provide more locally-grown food options for people in low-income areas. One way to do this would be to make inedible plants into a good source of nutrition and calories. Take cottonseed, for example, which has the potential to be a cheaper alternative to nuts. Cottonseeds are highly nutritious, containing oils and proteins in abundance, but many low-income cotton farmers cant eat cottonseeds because they produce toxins called gossypol.

Now, scientists have engineered cotton plants to remove the toxin, making cottonseeds safe for us to eat. And recently, the U.S. Food and Drug Administration approved genetically modified (GM) cottonseed for human consumption. Biotech cottonseed can act as an excellent alternative dietary source in low-income regions, where people struggle to meet the costs of the ideal diet recommended by EAT-Lancet.

Genetic engineering can also enable widespread cultivation of local plants. The groundcherry plant in its native form has a wild, sprawling growth habit which causes its fruits to drop to the ground while still small. Difficulties in cultivating the wildcherry mean its an orphan plant. However, scientists used genetic engineering to improve wildcherrys undesirable traits, including the plants weedy shape, flower production and fruit size. Now there are hopes for large-scale cultivation of genetically engineered groundcherry, which is native to Central and South America.

Millions of children and adults around the world suffer from micronutrient deficiencies, and biotechnology can also help fortify current crops to improve their vitamin and micronutrient contents. For example, scientists have recently developed biofortified cassava, which has higher zinc and iron contents than regular cassava. The biofortified cassava may one day prevent illnesses related to iron and zinc deficiencies.

Golden Rice is perhaps the prime example of a biofortified cropconventional rice that is genetically engineered to produce the vitamin A precursor beta-carotene. Golden Rice, acting as a source of vitamin A, can address vitamin A deficiency that blinds and kills hundreds of thousands of children every year. After a rigorous biosafety assessment in the Philippines, the Department of Agriculture-Bureau of Plant Industry found Golden Rice to be safe as conventional rice. Golden Rice regulation application is under review in Bangladesh, as well. This biofortified crop can provide much-needed micronutrients, taking the everyday staple food further to meet peoples dietary requirements in the poorest regions of the world.

Economic benefits

Improved agrobiodiversity and availability of local food varieties, enabled by biotechnology, will bring down the cost of the ideal diet, reducing food inequality. But GM technology also has the power to lift people out of poverty and increase the spending power of the low-income communities in developing regions.

Higher farm productivity, especially in low-income areas, can lower food prices. A meta-analysis of studies published after 1995 found that adopting GM technology has widespread benefits, including economic gains for farmers that grow GM crops. The meta-analysis found that GM technology increases crop yields by 21 percent. Some GM crops are engineered to be more resistant to pest damage, which helps achieve higher yields, for example.

The meta-study also found that GM crops require 37 percent less pesticide, which reduces pesticide costs by 39 percent and helps spare the environment. Even though GM seeds are more expensive than non-GM seeds, savings in pest control and pesticide use mean that farmers adopting GM crops enjoy 68 percent more profit. Therefore, GM crops can increase farmers spending power, which is excellent news for the quarter of the worlds working population employed in agriculture . More importantly, the yield and profit from GM crops are higher in developing countries than in developed countries.

If adopted widely, genetic engineering technology will bring us closer to meeting the EAT-Lancet dietary targets, which will help us protect the environment, public health, and reduce inequality.

Rupesh Paudyal holds a PhD in plant science and covers agriculture and the environment as a freelance writer. Visit his website and follow him on Twitter @TalkPlant

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Viewpoint: We can sustainably feed 10 billion people. Here's how CRISPR and GMO crops can help - Genetic Literacy Project

Traditional agriculture requires many inputs; fertilizer, specific chemicals, manual labor and water. Most of the water used in agriculture is for irrigation, and some crops require more water to grow than others. Rice is one of the most water-intensive crops, and also one of the most widely consumed worldwide.

Manipulating the rice genome is not entirely new. The Golden Rice Project emerged in 1999 to address the rampant vitamin A deficiency, and resulting blindness in manycountries where rice is a staple food. Other research into increasing photosynthetic efficiency, drought resistance, and methane reduction of rice is in the works as well, and all requires genetic modification.

Opposition to genetically modified organisms (GMOs) in food has halted progress on a project that the founders believe could save billions of people who eat rice every day. GMO use is a divisive topic, and many scientists and companies are choosing to stay away from them to avoid public disdain and regulatory challenges.

Agrisea is taking a different approach to food science. They want to grow rice in the ocean by using gene-editing, which would amplify the expression of genes already found in rice that control salt-tolerance. Salt-tolerant rice could be grown in salty ocean water without the use of soil, fertilizer or fresh water. Rather than inserting genes from other species, they have identified the genes that control for salt expulsion, cellular insulation and DNA protection, and are enhancing the expression of those genes.

Together these genes act in a network, just like they do in nature, Luke Young, CEO and co-founder of Agrisea said. We just encourage them along the pathways that nature has formed in plants that can thrive in a salty environment. The co-founders explained that they could use repeated selective breeding in rice to get the same result, but gene-editing just speeds up the process.

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Gene editing 'rice revolution': CRISPR could be used to grow one of the world's most important crops in salt water - Genetic Literacy Project

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CRISPR and CAS Gene Market Demand Analysis by 2026 | Caribou Biosciences Inc., CRISPR Therapeutics, Mirus Bio LLC, Editas Medicine, Takara Bio Inc.,...

Nearly 1.8 million Americans were diagnosed with cancer last year. Around the world, the total was close to 17 million. It's not surprising, then, that more than 700 biopharmaceutical companies have experimental cancer drugs in late-stage development.

Beta-thalassemia, on the other hand, is a rare disease that affects around 1,000 or so people in the United States. It's more prevalent in some countries but still impacts only one in 100,000 individuals.

You might expect one or maybe two biotechs could be developing therapies to treat beta-thalassemia. However, by my count, at least half a dozen companies have programs targeting the blood disorder. Why are a disproportionate number of biotechs scrambling to develop drugs for the same rare disease?

Image source: Getty Images.

Probably the main reason why a relatively large group of drugmakers are targeting beta-thalassemia is that the cause of the disease is straightforward. Understanding the why behind a disease is a critical prerequisite to treating it.

Beta-thalassemia is usually caused by a mutation in the HBB gene, which provides instructions on how to build beta-globin proteins. These proteins are part of hemoglobin, the protein in red blood cells that carries oxygen throughout the body. The HBB mutations that cause beta-thalassemia result in dysfunctional red blood cells that can't carry enough oxygen, which leads to patients experiencing anemia.

Another potential reason why biotechs are attracted to beta-thalassemia, though, is that it's not the only disease that is caused by mutations in the HBB gene. Sickle cell disease (SCD) is a related disease where HBB mutations cause red blood cells to form a sickle (or crescent) shape. These misshaped red blood cells can get stuck in blood vessels and cause multiple health complications, including anemia, infections, frequent pain, and heart problems.

While beta-thalassemia is rare, SCD is the most common genetic blood disorder in the U.S. It affects up to 100,000 Americans. SCD is even more prevalent in Africa, impacting up to 3% of newborns in some parts of the continent.

Drugmakers that identify a way to treat beta-thalassemia can be on the right track to target sickle cell disease as well. And with a much larger patient population, the market potential for successful therapies is greater.

One product has already been approved by the FDA for treating beta-thalassemia. Acceleron Pharma (NASDAQ:XLRN) developed luspatercept in collaboration with Celgene. In November 2019, Celgene won FDA approval for luspatercept in treating transfusion-dependent beta-thalassemia. Bristol-Myers Squibb (NYSE:BMY) closed its acquisition of Celgene a few weeks later and is marketing the drug under the brand name Reblozyl. Luspatercept is also in a mid-stage clinical study for treating non-transfusion-dependent beta-thalassemia.

Bluebird bio (NASDAQ:BLUE) won European approval for Lentiglobin in June 2019 for treating transfusion-dependent beta-thalassemia. Lentiglobin is a gene therapy that transplants cells with healthy HBB genes into patients. The biotech launched the therapy in Germany in January with the brand name Zynteglo. Bluebird plans to roll out Zynteglo in other key European markets later this year and should file for U.S. approval within the next few months.

Several biotechs are developing gene-editing approaches to treat beta-thalassemia. The company with the most advanced gene-editing program is Sangamo Therapeutics (NASDAQ:SGMO). However, there are some worries about ST-400, the experimental gene therapy that Sangamo is developing with Sanofi. In December 2019, Sangamo announced preliminary results from an early stage clinical study that, while showing promise, raised safety concerns.

CRISPR Therapeutics (NASDAQ:CRSP) and its big partner, Vertex Pharmaceuticals (NASDAQ:VRTX), are evaluating CTX001 in early stage clinical studies for treating beta-thalassemia and SCD. CTX-001 uses CRISPR gene editing, a different method than the zinc-finger nuclease (ZFN) gene-editing approach that Sangamo uses. CRISPR Therapeutics and Vertex reported promising preliminary results in December 2019 from both of its clinical studies.

Editas Medicine (NASDAQ:EDIT) is also using CRISPR gene editing to target both beta-thalassemia and SCD. The biotech hasn't advanced its experimental therapy to a clinical study in humans yet but plans to file for FDA approval later in 2020 to begin clinical testing. Editas thinks that its gene-editing approach is superior to the ones being taken by CRISPR Therapeutics and Sangamo.

Trailing the pack is Syros Pharmaceuticals (NASDAQ:SYRS). In December, Syros and Global Blood Therapeuticssigned a deal to work together to develop drugs targeting beta-thalassemia and SCD based on Syros' gene control platform. Instead of trying to directly edit the gene mutations, Syros' gene control therapies attempt to control the expression of genes through genomic switches in other parts of DNA. The biotech hasn't said how soon it will be able to advance to clinical testing with its experimental drug.

There are a couple of big problems for investors with so many companies chasing after the same rare disease. First, it's impossible to know which experimental therapies will be successful. Second, if multiple drugs win regulatory approvals, the competition could be so fierce that no product is a huge moneymaker.

It's also important to know that several of the products being developed hold the potential to cure beta-thalassemia. These therapies could wipe out the opportunities for drugs that aren't curative.

One solution to this investor's dilemma is to avoid all of the biotech stocks that are focused on beta-thalassemia. However, that's like throwing the baby out with the bathwater. I think that a better alternative is to invest in the big drugmakers with beta-thalassemia programs.

Bristol-Myers Squibb already has one FDA approval under its belt for Reblozyl. BMS also owns 5.3% of CRISPR Therapeutics and is partnering with Editas on developing gene-editing therapies targeting cancer. Vertex is partnering with CRISPR Therapeutics and owns 10.2% of the small biotech. Both BMS and Vertex stand to win with their beta-thalassemia drugs but also have plenty of other growth drivers.

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Why So Many Biotechs Are Scrambling to Develop a Drug for the Same Rare Disease - The Motley Fool

The yellow monkeyflowers distinctive red spots serve as landing pads for bees and other pollinators, helping them access the sweet nectar inside. A new study reveals the genetic programming that creates these attractive patterns. (Image byPollyDotviaPixaBay)

February 24, 2020 - ByKara Manke- The intricate spotted patterns dappling the bright blooms of the monkeyflower plant may be a delight to humans, but they also serve a key function for the plant. These patterns act as bee landing pads, attracting nearby pollinators to the flower and signaling the best approach to access the sweet nectar inside.

They are like runway landing lights, helping the bees orient so they come in right side up instead of upside down, said Benjamin Blackman, assistant professor of plant and molecular biology at the University of California, Berkeley.

In a new paper, Blackman and his group at UC Berkeley, in collaboration with Yaowu Yuan and his group at the University of Connecticut, reveal for the first time the genetic programming that helps the monkeyflower and likely other patterned flowers achieve their spotted glory. Thestudywas published online today (Thursday, Feb. 20) in the journalCurrent Biology.

While we know a good deal about how hue is specified in flower petals whether it is red or orange or blue, for instance we dont know a lot about how those pigments are then painted into patterns on petals during development to give rise to these spots and stripes that are often critical for interacting with pollinators, Blackman said. Our lab, in collaboration with others, has developed the genetic tools to be able to identify the genes related to these patterns and perturb them so that we can confirm whats actually going on.

In the study, the research team used CRISPR-Cas9 gene editing to recreate the yellow monkeyflower patterns found in nature. On the left, a wild-type monkeyflower exhibits the typical spotted pattern. In the middle, a heterozygote with one normal RTO gene and one damaged RTO gene exhibits blotchier spots. And on the right, homozygote with two copies of the damaged RTO gene is all red, with no spots. (UC Berkeley photo by Srinidhi Holalu)

The positions of petals spots arent mapped out ahead of time, like submarines in a game of battleship, Blackman said. Instead, scientists have long theorized that they could come about through the workings of an activator-repressor system, following what is known as a reaction-diffusion model, in which an activator molecule stimulates a cell to produce the red-colored pigment that produces a spot. At the same time, a repressor molecule is expressed and sent to neighboring cells to instruct themnotto produce the red pigment.

The results are small, dispersed bunches of red cells surrounded by cells that keep the background yellow color.

By tweaking the parameters how strongly a cell turns on an inhibitor, how strongly the inhibitor can inhibit the activator, how quickly it moves between cells it can lead to big spots, small spots, striped patterns, really interesting periodic patterns, Blackman said.

In the study, UC Berkeley postdoctoral researcher Srinidhi Holalu and research associate Erin Patterson identified two natural varieties of the yellow monkeyflower one type with the typical red spots in the throat of the flower and a second type with an all-red throat appearing in multiple natural populations in California and Oregon, including at theUC Davis McLaughlin Reserve. In parallel, UConn postdoctoral researcher Baoqing Ding worked with a very similar plant with fully red-throated flowers found when surveying a population of Lewiss monkeyflower that had induced DNA mutations.

When the scientists presented bees in the lab with the two types of monkeyflowers, they preferred the red tongue variety to the spotted variety, though the red tongue variety is less common in nature. (UC Berkeley video by Erin Patterson and Anna Greenlee)

In a previous study, the Yuan lab had found that a gene called NEGAN (nectar guide anthocyanin) acts as an activator in the monkeyflower petals, signaling the cells to produce the red pigment. Through detailed genomic analysis in both monkeyflower species, the two groups were able to pinpoint that a gene called RTO, short for red tongue, acts as the inhibitor.

The red-throated forms of the monkeyflower have defective RTO inhibitor genes, resulting in a characteristic all-red throat, rather than red spots. To confirm their findings, Holalu used the CRISPR-Cas9 gene editing system to knock out the RTO gene in spotted variants of the flower. The result was flowers with a flashy red throat. Further experiments revealed how the functional form of the RTO protein moves to neighboring cells and represses NEGAN to prevent the spread of pigmentation beyond the local spots. This study is the first reported use of CRISPR-Cas9 editing to research the biology of monkeyflowers.

The team also collaborated with Michael Blinov at the UConn School of Medicine to develop a mathematical model to explain how different self-organized patterns might arise from this genetic system.

This work is the simplest demonstration of the reaction-diffusion theory of how patterns arise in biological systems, said Yaowu Yuan, associate professor of ecology and evolutionary biology at UConn. We are closer to understanding how these patterns arise throughout nature.

Monkeyflower plants with the RTO gene knocked out by CRISPR-Cas9 gene editing produce one big patch where all flowers exhibit a fully red throat, in contrast to wild fields where red-tongued flowers appear in small dispersed spots (UC Berkeley photo by Srinidhi Holalu)Source: UC Berkeley

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UC Professor On How The Monkeyflower Gets Its Spots - Sierra Sun Times

CHAMPAIGN, Ill. With a new CRISPR gene-editing methodology, scientists from the University of Illinois at Urbana-Champaign inactivated one of the genes responsible for an inherited form of amyotrophic lateral sclerosis a debilitating and fatal neurological disease for which there is no cure. The novel treatment slowed disease progression, improved muscle function and extended lifespan in mice with an aggressive form of ALS.

ALS unfortunately has few treatment options. This is an important first step in showing that this new form of gene editing could be used to potentially treat the disease, said bioengineering professor Thomas Gaj, who co-led the study with bioengineering professor Pablo Perez-Pinera.

The method relied on an emerging gene-editing technology known as CRISPR base editors.

Traditional CRISPR gene-editing technologies cut both strands of a DNA molecule, which can introduce a variety of errors in the DNA sequence, limiting its efficiency and potentially leading to a number of unintended mutations in the genome. The Illinois group instead used base editing to change one letter of the DNA sequence to another without cutting through both DNA strands, Perez-Pinera said.

Base editors are too large to be delivered into cells with one of the most promising and successful gene therapy vectors, known as adeno-associated virus, Gaj said. However, in 2019, Perez-Pineras group developed a method of splitting the base editor proteins into halves that can be delivered by two separate AAV particles. Once inside the cell, the halves reassemble into the full-length base editor protein.

By combining the power of AAV gene delivery and split-base editors, Gaj and Perez-Pinera targeted and permanently disabled a mutant SOD1 gene, which is responsible for roughly 20% of inherited forms of ALS. They published their results in the journal Molecular Therapy.

Many ALS studies are focused on preventing or delaying the onset of the disease. However, in the real world, most patients are not diagnosed until symptoms are advanced, said graduate student Colin Lim. Slowing progression, rather than preventing it, may have a greater impact on patients. Lim is the co-first author of the study along with graduate students Michael Gapinske and Alexandra Brooks.

CRISPR base editing decreased the amount of a mutant protein (blue) that contributes to ALS in the spinal cord. Left, a spinal cord section from an untreated mouse. Right, a spinal cord section from an animal treated by base editing.

Image courtesy of Thomas Gaj

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The researchers first tested the SOD1 base editor in human cells to verify reassembly of the split CRISPR base editor and inactivation of the SOD1 gene. Then they injected AAV particles encoding the base editors into the spinal columns of mice carrying a mutant SOD1 gene that causes a particularly severe form of ALS that paralyzes the mice within a few months after birth.

The disease progressed more slowly in treated mice, which had improved motor function, greater muscle strength and less weight loss. The researchers observed an 85% increase in time between the onset of the late stage of the disease and the end stage, as well as increased overall survival.

We were excited to find that many of the improvements happened well after the onset of the disease. This told us that we were slowing the progression of the disorder, Gapinske said.

The base editor introduces a stop signal near the start of the SOD1 gene, so it has the advantage of stopping the cell from making the malfunctioning protein no matter which genetic mutation a patient has. However, it potentially disrupts the healthy version of the gene, so the researchers are exploring ways to target the genes mutant copy.

Moving forward, we are thinking about how we can bring this and other gene-editing technologies to the clinic so that we can someday treat ALS in patients, Gaj said. For that, we have to develop new strategies capable of targeting all of the cells involved in the disease. We also have to further evaluate the efficiency and safety of this approach in other clinically relevant models.

The split base editor approach has potential for treating other diseases with a genetic basis as well, Perez-Pinera said. Though ALS was the first demonstration of the tool, his group has studies underway applying it to Duchenne muscular dystrophy and spinal muscular atrophy.

The Muscular Dystrophy Association, the Judith and Jean Pape Adams Foundation, the American Heart Association and the National Institutes of Health supported this work. Gaj and Perez-Pinera are affiliated with the Carl R. Woese Institute for Genomic Biology at Illinois. Perez-Pinera also is affiliated with the Carle Illinois College of Medicine and the Cancer Center at Illinois.

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New CRISPR base-editing technology slows ALS progression in mice - University of Illinois News