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Archive for the ‘Crispr’ Category

Addgene: CRISPR References and Information

This table lists gRNA sequences that have been experimentally validated for use in CRISPR experiments.

gRNA design tool with extensive selection of eukaryotic pathogen genomes (200+) that can predict gRNA targets in gene families, HDR oligonucleotide design, and batch processing for designing genome-wide gRNA libraries. PubMed PMID 28348817.

This tool helps design (10 different prediction scores), clone (primer design), and evaluate gRNAs, as well as predict off-targets, for CRISPR in 180+ genomes. PubMed PMID: 27380939.

sgRNA Scorer 2.0From the Church Lab: a tool that identifies putative target sites for S. pyogenes Cas9, S. thermophilus Cas9, or Cpf from your input sequence or list of sequences.

Quilt Universal guide RNA designerSearch for gRNAs via gene name or by genomic location. Database includes gRNAs from popular CRISPR libraries and from more than two million DNAse hypersensitive sites for intergenic guide RNAs in hg19, filtered for off-target effects.

From the Kim Lab, Cas-OFFinder identifies gRNA target sequences from an input sequence and checks for off-target binding. Currently supports: Drosophila, Arabidopsis, zebrafish, C. elegans, mouse, human, rat, cow, dog, pig, Thale cress, rice (Oryza sativa), tomato, corn, monkey (macaca mulatta).

Cas-Designer searches for targets that maximize knockout efficiency while having a a low probability of off-target effects. Cas-Designer integrates information from the Kim Lab’s Cas-OFFinder and Microhomology predictor.

From the Qi Lab, a sgRNA design tool for genome editing, as well as gene regulation (repression and activation). Genome support for bacteria (E. coli, B. subtilis), yeast (S. cerevisiae), worm (C. elegans), fruit fly, zebrafish, mouse, rat, and human.

Identifies candidate sgRNA target sites by off-target quality. Validated for gene inactivation, NHEJ, and HDR. Reference genomes include Arabidopsis, C. elegans , sea squirt, cavefish, Chinese hamster, fruit fly, human, rice fish, mouse, silk worm, stickleback, tobacco, tomato, frog (X. laevis and X. tropicalis), and zebrafish.

Program for designing optimal gRNAs. Provides feedback on number of potential off-targets, target’s genomic location, and genome annotation. Available genomes are human (hg19 & hg38), mouse (mm10), and yeast (strain w303).

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Addgene: CRISPR References and Information

Here’s Why CRISPR Therapeutics Lost 18.8% in July — The …

What happened

Shares of CRISPR Therapeutics (NASDAQ:CRSP) fell nearly 19% last month, according to data provided by S&P Global Market Intelligence, after yet another study reminded Wall Street and investors that there’s still much for scientists to understand about the use of CRISPR gene-editing tools in human cells. Previously, in June, two studies surfaced that suggested certain uses of CRISPR could trigger faulty DNA repair mechanisms to activate and turn a cell cancerous.

That was followed up last month by a new study suggesting that certain uses of CRISPR tools “seriously underestimated” the number of off-target changes made to a genome. CRISPR Therapeuticssaid to Reutersthat “[w]e do not use the methods described in this Nature Biotech paper … nevertheless, in our work, we do not see similar findings.” While that wasn’t enough to appease Wall Street in July, shareholders have still enjoyed a year-to-date gain of 103%.

Image source: Getty Images.

The study published last month came from researchers at the prestigious Wellcome Sanger Institute, an affiliation that helped the results to be taken more seriously. But considering CRISPR Therapeutics says it doesn’t use the specific techniques identified, investors may be wondering why the company’s shares were impacted at all. Well, it has to do with increasing uncertainty over an important part of using certain gene-editing tools.

More specifically, the most recent study detailing off-target changes to DNA and those identifying the potential to activate cancerous mutations already present in cells all seem to imply the same thing: Scientists may have gotten a little ahead of themselves by assuming DNA repair mechanisms would work in a simple fashion. While CRISPR tools intend to fix genetic defects by cutting one or both strands of human DNA, all rely on DNA repair mechanisms already present in a cell to stitch the genome back together. If those fail, then CRISPR tools might be less effective or could even end up having significant unintended effects.

Right now, it appears that the most troubling side effects are observed when CRISPR tools cut both strands of DNA (a “double-strand break”). The lead drug candidates of all three major CRISPR companies deploying the technology for medical applications avoid that headache, although all companies are exploring preclinical therapeutics that will have to navigate that obstacle eventually.

Investors can likely expect gene-editing stocks such as CRISPR Therapeutics to experience a higher-than-normal amount of volatility. The technology has received an incredible amount of attention in the media and even popular culture, and the potential to cure diseases has Wall Street understandably excited. Those forces have combined to hand the pioneering companies premium market valuations, but it’s important to remember that CRISPR is a relatively new technology. Investors in it for the long haul will simply need to buckle up and remain patient as results from the first clinical trials (yet to get started) begin to trickle in within the next few years.

Maxx Chatsko has no position in any of the stocks mentioned. The Motley Fool owns shares of CRISPR Therapeutics. The Motley Fool has a disclosure policy.

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Here’s Why CRISPR Therapeutics Lost 18.8% in July — The …

Here’s Why CRISPR Stocks Are Down as Much as 11.4% Today …

What happened

Shares of the three leading companies developing human therapeutics based on CRISPR gene-editing technology fell as much as 11.4% today. There was no new news that could be interpreted as detrimental to CRISPR Therapeutics (NASDAQ:CRSP), Editas Medicine (NASDAQ:EDIT), or Intellia Therapeutics (NASDAQ:NTLA). But on July 23, many media outlets published stories commenting on a study released one week earlier.

While the stock moves may seem to be based on the rehashing of old news, there are good reasons investors shouldn’t be too quick to dismiss the concerns. As of 2:44 p.m. EDT, CRISPR Therapeutics stock had settled to a 9.5% loss, while Editas Medicine shares were down 10.4%, and Intellia Therapeutics stock had sunk by 7.2%.

Image source: Getty Images.

On July 16, scientists published a study in Nature Biotechnology demonstrating that using CRISPR tools to edit faulty DNA sequences can lead to unintended deletions and rearrangements of genetic material. The lead author, Dr. Allan Bradley, issued a cautious summary of the study:

This is the first systematic assessment of unexpected events resulting from CRISPR/Cas9 editing in therapeutically relevant cells, and we found that changes in the DNA have been seriously underestimated before now. It is important that anyone thinking of using this technology for gene therapy proceeds with caution, and looks very carefully to check for possible harmful effects.

The researchers, who hail from the prestigious Wellcome Sanger Institute, found that some of the genetic changes occurred far away from where CRISPR tools cut a genome, locations which would elude existing diagnostic tools used to gauge off-target effects. In other words, the field has “seriously underestimated” the potential for unintended genetic alterations because it hasn’t been looking in the right places.

All three companies made statements to Reuters last week regarding the study. CRISPR Therapeutics commented: “We do not use the methods described in this Nature Biotech paper … nevertheless, in our work, we do not see similar findings.” Editas Medicine said it was “not specifically concerned.” Intellia Therapeutics said it didn’t think the findings would affect the future of CRISPR-based therapies.

While it’s important for investors not to panic over the latest study showing potentially unintended consequences of using gene-editing tools, it is worth noting that most of the recent uncertainty injected into CRISPR stocks has come from observations of DNA repair mechanisms — one thing gene-editing tools have little to no control over. While companies focus on developing safe and effective ways to cut a genome, they must rely on natural cellular processes to stitch up the genome afterwards.

For instance, in June, investors worried over two studies showing that CRISPR tools could activate a faulty DNA repair mechanism and result in cancerous cells. The latest study from the Wellcome Sanger Institute was not concerned with the same question, but demonstrated that researchers may be overlooking the details of how genomes get stitched back up.

An open-minded approach to investing in CRISPR Therapeutics, Editas Medicine, and Intellia Therapeutics would nod to the awesome potential of the technology while acknowledging the risks of an early-stage investment. Recent stock moves hint that the hype may need to come back down to earth, as there’s much left to understand about using CRISPR tools in human cells. To date researchers have focused mostly on the ability to cut DNA, but it may be time to start paying closer attention to what happens after that.

Maxx Chatsko has no position in any of the stocks mentioned. The Motley Fool owns shares of CRISPR Therapeutics. The Motley Fool recommends Editas Medicine. The Motley Fool has a disclosure policy.

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Here’s Why CRISPR Stocks Are Down as Much as 11.4% Today …

Our Programs – CRISPR

Gene Editing to Treat Disease

The majority of medical therapies available today are directed at managing disease processes, the pathogenic or mis-regulated proteins or molecules associated with disease. However, these pathogenic molecules themselves are typically encoded in or affected by changes in genes or other sequences in the human genome, which encompasses the DNA in all our cells. Gene editing technologies, including CRISPR/Cas9, now offer us the ability to directly modify or correct the underlying disease-associated changes in our genome. Successfully editing or correcting a gene that encodes the dysfunctional or missing protein can in principle result in the expression of a fully normal protein and full correction of the disease.

Gene therapy and other technologies to modify the genome have been in development for many years, and a small number of gene therapies have been approved to treat patients. However, these older approaches have been burdened by challenges to their safety and efficacy and have not yet provided the ability to precisely control a range of different genetic changes.

We believe that CRISPR/Cas9 offers just such an opportunity, particularly to correct DNA changes in somatic (non germ line) cells in patients with serious disease.

CRISPR/Cas9 is a rapid and easy to use gene editing technology that can selectively delete, modify or correct a disease causing abnormality in a specific DNA segment. CRISPR refers to Clustered Regularly Interspaced Short Palindromic Repeats occurring in the genome of certain bacteria, from which the system was discovered; Cas9 is a CRISPR-associated endonuclease (an enzyme), the molecular scissors that are easily programmed to cut and edit, or correct, disease-associated DNA in a patients cell. The location at which the Cas9 molecular scissors cut the DNA to be edited is specified by guide RNA, which is comprised of a crRNA component and a tracrRNA component, either individually or combined together as a single guide RNA (sgRNA). For example, a guide RNA can direct the molecular scissors to cut the DNA at the exact site of the mutation present in the genome of patients with a particular genetic disease. Once the molecular scissors make a cut in the DNA, additional cellular mechanisms and exogenously added DNA will use the cells own machinery and other elements to specifically repair the cut DNA.

There are more than 10,000 known single-gene (or monogenic) diseases, occurring in about 1 out of every 100 births1. Scientists and clinicians are now conducting pioneering research using CRISPR/Cas9 to address both recessive and dominant genetic defects, opening up the potential of gene editing to provide novel transformative gene-based medicines for patients with a large number of both rare and common diseases.

Dr. Emmanuelle Charpentier, one of CRISPR Therapeutics scientific founders, co-invented the CRISPR/Cas9 technology.

The clustered repeats of CRISPR were discovered in 1987 in bacteria2, but their function was unknown. In 2000, these clustered repeat elements were found to be relatively common in bacteria3 hinting to an important role of these elements. The clustered repeats were given the name CRISPR in 2002 and multiple CRISPR-associated (Cas) genes were discovered adjacent to the repeat elements in that same year4.

CRISPR/Cas9 is a rapid and easy to use gene editing technology that can selectively delete, modify or correct a disease causing abnormality in a specific DNA segment. CRISPR refers to Clustered Regularly Interspaced Short Palindromic Repeats occurring in the genome of certain bacteria, from which the system was discovered; Cas9 is a CRISPR-associated endonuclease (an enzyme), the molecular scissors that are easily programmed to cut and edit, or correct, disease-associated DNA in a patients cell. The location at which the Cas9 molecular scissors cut the DNA to be edited is specified by guide RNA, which is comprised of a crRNA component and a tracrRNA component, either individually or combined together as a single guide RNA (sgRNA). For example, a guide RNA can direct the molecular scissors to cut the DNA at the exact site of the mutation present in the genome of patients with a particular genetic disease. Once the molecular scissors make a cut in the DNA, additional cellular mechanisms and exogenously added DNA will use the cells own machinery and other elements to specifically repair the cut DNA.

There are more than 10,000 known single-gene (or monogenic) diseases, occurring in about 1 out of every 100 births1. Scientists and clinicians are now conducting pioneering research using CRISPR/Cas9 to address both recessive and dominant genetic defects, opening up the potential of gene editing to provide novel transformative gene-based medicines for patients with a large number of both rare and common diseases.

Dr. Emmanuelle Charpentier, one of CRISPR Therapeutics scientific founders, co-invented the CRISPR/Cas9 technology.

The clustered repeats of CRISPR were discovered in 1987 in bacteria2, but their function was unknown. In 2000, these clustered repeat elements were found to be relatively common in bacteria3 hinting to an important role of these elements. The clustered repeats were given the name CRISPR in 2002 and multiple CRISPR-associated (Cas) genes were discovered adjacent to the repeat elements in that same year4.

The function of the CRISPR-Cas system in bacteria as an immune defense mechanism was hypothesized by Mojica in 20055 and experimentally validated at the food ingredient company, Danisco, in 20076.

In 2011, Dr. Charpentiers lab discovered an essential component of the CRISPR-Cas system, tracrRNA, in bacteria7. The following year she and colleagues described how the Cas9 endonuclease works together with crRNA and tracrRNA to form functional molecular scissors to cut at a specific DNA sequence in the genome8. In this same publication the authors also described how to modify, or re-program, the system to direct the molecular scissors to cut at essentially any DNA sequence; how to modify the RNA components into a single guide RNA, simplifying the system into only 2 components; and how to modify the Cas9 molecular scissors to make nicks in the DNA by only cutting one of the two DNA strands. These foundational discoveries enabled transformative gene editing in a wide range of cells, tissues and species, including for the potential benefit of patients suffering from serious genetic diseases.

CRISPR/Cas9 is an easy, effective technology for gene editing that has enabled a wide range of new studies and transformed many areas of research. Thousands of academic laboratories across the world are carrying out research using the technology. Rapid adoption of CRISPR/Cas9 by the broader academic community and the collective efforts of their research are in turn driving tremendous progress in the field.

We have licensed the foundational CRISPR/Cas9 patent estate for human therapeutic use from our scientific founder, Dr. Emmanuelle Charpentier. This IP is directed broadly to CRISPR/Cas9 genome editing and includes many different applications of the technology. We have filed additional IP and will continue to do so in support of our mission to bring transformative gene-based medicines to patients with serious diseases.

In 2011, Dr. Charpentiers lab discovered an essential component of the CRISPR-Cas system, tracrRNA, in bacteria7. The following year she and colleagues described how the Cas9 endonuclease works together with crRNA and tracrRNA to form functional molecular scissors to cut at a specific DNA sequence in the genome8. In this same publication the authors also described how to modify, or re-program, the system to direct the molecular scissors to cut at essentially any DNA sequence; how to modify the RNA components into a single guide RNA, simplifying the system into only 2 components; and how to modify the Cas9 molecular scissors to make nicks in the DNA by only cutting one of the two DNA strands. These foundational discoveries enabled transformative gene editing in a wide range of cells, tissues and species, including for the potential benefit of patients suffering from serious genetic diseases.

CRISPR/Cas9 is an easy, effective technology for gene editing that has enabled a wide range of new studies and transformed many areas of research. Thousands of academic laboratories across the world are carrying out research using the technology. Rapid adoption of CRISPR/Cas9 by the broader academic community and the collective efforts of their research are in turn driving tremendous progress in the field.

We have licensed the foundational CRISPR/Cas9 patent estate for human therapeutic use from our scientific founder, Dr. Emmanuelle Charpentier. This IP is directed broadly to CRISPR/Cas9 genome editing and includes many different applications of the technology. We have filed additional IP and will continue to do so in support of our mission to bring transformative gene-based medicines to patients with serious diseases.

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Our Programs – CRISPR

Here’s Why CRISPR Stocks Have Gained as Much as 169% in 2018 …

What happened

Shares of the three companies pioneering medical applications of CRISPR gene-editing tools have soared higher through the first half of 2018. That’s because, after years of only being able to discuss the possibilities of the technology, investors will soon be able to watch it (hopefully) progress through regulated clinical trials.

According to data from S&P Global Market Intelligence, CRISPR Therapeuticstops the trio with year-to-date gains of 169% and a market cap of nearly $3 billion. Intellia Therapeuticsis next with a 63% rise and a market cap of $1.3 billion. Editas Medicine, which has managed a 25% leap since the beginning of the year, is valued at $1.8 billion. It entered January as the most valuable of the three.

Image source: Getty Images.

All three companies are about to investigate their unique CRISPR tools in the clinic for the first time, and their respective stock performances thus far this year correlate with how close each is to initiating clinical trials.

CRISPR Therapeutics is the furthest along, looking to begin clinical trials for its lead drug candidate, CTX001, as a treatment for blood diseases such as sickle cell by the end of this year. While it was placed on a clinical hold by the U.S. Food and Drug Administration at the end of May, the company and its partner Vertex will proceed with a phase 1/2 trial in Europe as planned. Not wanting to rest on its lead, the company is looking to file its second investigational new drug (IND) application by the end of 2018.

Meanwhile, Editas Medicine told investors it would file an IND for its lead drug candidate in mid-2018, so investors should expect that news any day now. The company will first take aim at LCA10, a rare eye disease.

Intellia Therapeutics is furthest behind, as it doesn’t expect to file an IND until the end of 2019. That could work out in the company’s favor in the long run, however, as it’s working on a novel delivery system (one of the biggest question marks for all three companies) to increase the efficacy and safety of its therapeutics, the first of which will be evaluated to treat a rare metabolic disease called transthyretin amyloidosis.

Company

End Q1 2018 Cash Balance

IND Filing Guidance

CRISPR Therapeutics (NASDAQ:CRSP)

$342 million

Initiating first clinical trial by end of 2018, second IND by end of 2018

Editas Medicine (NASDAQ:EDIT)

$359 million

Mid-2018

Intellia Therapeutics (NASDAQ:NTLA)

$328 million

End of 2019

Data source: Company disclosures.

All three stocks have largely brushed off concerns raised in June that CRISPR tools could potentially set off existing and potentially cancerous mutations within cells. While none of the lead drug candidates would be affected by the approaches being used, all three pipelines will have to navigate that obstacle eventually.

Investors are betting that gene editing will become a game changer in medicine — and they might be right. However, it’s important to remember that CRISPR tools are still in their infancy in the clinic. There are still questions about optimal delivery of the therapeutic payload into human cells in a patient, the best cutting enzyme, and the triggering of DNA repair mechanisms being relied on to finish the genetic surgery procedure. Each has implications for the efficacy and safety of the technology.

Considering these questions (and more) will find their first answers in clinical trials that have yet to begin, and the fact these companies are valued at up to $3 billion, investors should understand the high level of risk involved with CRISPR stocks at this point in development. There could be a long way to go before reality matches the hype.

Maxx Chatsko has no position in any of the stocks mentioned. The Motley Fool owns shares of CRISPR Therapeutics. The Motley Fool recommends Editas Medicine. The Motley Fool has a disclosure policy.

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CRISPR News – GenScript

CRISPR Plasmids

DNA plasmids for single guide RNA and/or Cas9 expression

Validated knock-out cell line service using CRISPR technology.

Genome-wide or pathway-specific CRISPR knock-out or activation libraries for screening experiments.

Validated knock-out and knock-in mutagenesis in bacteria and yeast.

CRISPR News

July 6, 2017

For the first time, researchers have been able to detect and characterize the mechanism of action by which the CRISPR complex binds and cleaves DNA using electron microscopy. Scientists at Harvard and Cornell have recently created near-atomic level resolution images of the CRISPR/Cas3 complex, a common CRISPR/Cas subtype, which provide structural data that can improve gene editing accuracy and efficiency.

To solve problems of specificity, we need to understand every step of CRISPR complex formation, states Maofu Liao, a co-author of the study and assistant professor at Harvard. Our study now shows the precise mechanism for how invading DNA is captured by CRISPR, from initial recognition of target DNA and through a process of conformational changes that make DNA accessible for final cleavage by Cas3.

This discovery uncovers a number of novel, overlapping mechanisms which prevent off-target site cleavage. In the CRISPR/Cas3 system, the assembled CRISPR complex first searches for a corresponding protospacer adjacent motif (PAM) sequence, which indicates a possible target site. Researchers discovered that as the CRISPR complex detects the PAM, it also bends DNA at a sharp angle, forcing a small portion to unwind. This allows an 11-nucleotide stretch of the CRISPR guide RNA to bind onto the target DNA, creating a seed bubble. The seed bubble acts as a fail-safe mechanism to check whether target DNA matches the guide RNA. If correctly matched, the bubble is enlarged and the remainder of the guide RNA binds onto the DNA forming an R-loop structure. Only once the full R-loop structure is formed does the Cas enzyme bind and cut the DNA in the non-target DNA strand. This study is the first to reveal the full sequence of events from seed bubble formation to R-loop formation.

Looking for an affordable and easy way to model disease in vivo?Interested in performing a genome-wide screen?Use CRISPR RNA/Cas9 Reagents or CRISPR Plasmids for high efficiency, customizable gene editing.

Xiao, Y. et al. Structure Basis for Directional R-loop Formation and Substrate Handover Mechanisms in Type I CRISPR-Cas System.Cell170, 48-60.e11 (2017).

June 28, 2017

Today, nearly 1 out of every 68 children born is diagnosed with autism spectrum disorder (ASD). Globally the disease is estimated to affect over 25 million people. And prevalence is expected to rise with ASD identifications doubling in the last decade.

ASD describes a variety of neurodevelopmental disorders which are often characterized by deficits in social communication and interaction, and restricted and repetitive behavior. While no specific causes for ASD have yet been found, a number of genetic and environmental risk factors have been identified. Most recently, a new study from Columbia Universitys Mailman School of Public Health, has discovered that prenatal fever increases autism risk by up to 40%.

Researchers monitored over 95,000 children born between 1999 and 2009. Of that population, 15,701 children were identified to have mothers reporting fever conditions during pregnancy. These children were found to have increased risk of ASD by 34%. Risk increase was highest, at 40%, when fever was reported during the second trimester. And ASD risk was increased by over 300% for the children of women reporting three or more fevers after the twelfth week of pregnancy.

Our results suggest a role for gestational maternal infection and innate immune responses to infection in the onset of at least some cases of autism spectrum disorder, states lead researcher, Associate Professor Mady Hornig. Additional studies are ongoing to determine the role of specific infectious agents in the development of ASD.

Hornig, M. et al. Prenatal fever and autism risk. Molecular Psychiatry (2017). doi:10.1038/mp.2017.119

June 22, 2017

Each year almost 200,000 people in the U.S. require emergency medical care for a serious allergic reaction. This number is expected to grow as food allergy incidence has increased by 50% in the last decade.

Allergies are caused from the hypersensitivity of the immune system to allergens in the environment. Recognition of these allergens triggers a T-cell-mediated immune response, producing cytokines which induce chronic inflammation and mucous hypersecretion.

In a recent study at the University of Queensland, Professor Ray Steptoe has been able to de-sensitize T-cells using a novel gene therapy treatment. Dr. Steptoes research team has engineered bone marrow stem cells to express transgenic allergen proteins. This effectively tricks the body into identifying the transgenic allergen as a self-antigen originating from within the body, leading to negative selection of any reacting T-cells. After treatment, the immune system is memory wiped alleviating airway inflammation and hyperreactivity.

Dr. Steptoe states that the eventual goal will be to devise a single-dose injectable therapeutic, which could replace the various short-term treatments that focus on alleviating allergy symptoms. Potential patents would be those individuals who are suffering from potentially lethal allergies or severe asthma.

AL-Kouba, J. et al. Allergen-encoding bone marrow transfer inactivates allergic T cell responses, alleviating airway inflammation. JCI Insight2, (2017).

June 15, 2017

Each year nearly 2 million people in the USA are infected by antibiotic-resistant bacteria. With antibiotic resistance on the rise, scientists have begun to turn to alternative antimicrobial treatments.

At the University of Wisconsin-Madison, scientists are developing a new probiotic “CRISPR pill” that is effective even against drug-resistant threats. Researchers from the lab of Jan-Peter Van Pijkeren have engineered bacteriophages expressing customized CRISPR guide RNA sequences. These CRISPR RNAs hijack the innate bacterial CRISPR immunity system present in infectious bacteria, causing them to self-destruct by creating lethal breaks in their own DNA. The bacteriophages are packaged in pill form in a mixture of probiotics, allowing them to survive the digestive tract until reaching the intestines.

By utilizing the innate immune system present in bacteria, the CRISPR pill bypasses the main mechanisms of antibiotic resistance. In addition, CRISPR pills may be superior to traditional antibiotics, because of their narrow targeting spectrum which can target specific bacterial species and strains. In contrast, broad-spectrum antibiotics kill off both “good” and “bad” bacteria. And overuse of traditional antibiotics has lead to the rising epidemic of antibiotic-resistant infections.

CRISPR and the CRISPR Associated system (Cas) is a powerful gene editing technology. Originally identified and characterized in bacteria, endogenous CRISPR systems act as an RNA-based defense mechanism against invading phage DNA.

CRISPR was adapted for genome editing in 2013 and has since been exploited for its ability to generate targeted double-stranded DNA breaks, which has revolutionized molecular biology protocols.1,2

This guide covers the basics of CRISPR experimental design and will prepare you to embark upon your own genome editing experiment.

Endogenous CRISPR systems fall into three categories type I, II and II. You can read more about these types in Makarova et al.3 Commercial CRISPR genome editing tools are adapted and simplified from endogenous type II systems and have the following components:

When gRNA and Cas9 are expressed together in a cell, a gRNA:Cas9 complex is recruited to the target DNA sequence, which is located immediately upstream of a motif called a protospacer adjacent motif (PAM).4 The PAM motif targeted by most commercial Cas9 enzymes is NGG (any nucleotide followed by two guanines).

Binding of the gRNA to target DNA occurs via complementary base-pairing between the genomic target sequence and the 20-nucleotide spacer on the gRNA. The Cas9 in the gRNA:Cas9 complex then cuts the genomic DNA, inducing a double-stranded break after the PAM sequence. Crucially, Cas9 cannot digest DNA unless bound to the gRNA, thus providing specificity to the system.

The editing process is completed by repairing the break using the endogenous Non-Homologous End Joining (NHEJ) pathway. While this DNA repair system is the most efficient repair pathway it is error prone, sometimes permitting small insertions or deletions, which can result in frameshifts and reduced protein production. An alternative option is to exploit the endogenous Homology Directed Repair (HDR) system by providing the HR template, as mentioned above. This is used when introducing targeted mutations.

Once you have designed and cloned the gRNA and HR templates, you cotransfect the Cas9 plasmid and your gRNA and HR donor vectors into the chosen cell line. Lipid transfection, electroporation or microinjection are all suitable transfection methods.

Optimizing recombination levels may take some trial and error. Choose a robust cell line (e.g., HEK) for troubleshooting. Once your experiment is up and running, you can move onto more expensive and less robust cell lines, if necessary.

Bear in mind that immortalized cell lines are not only cheaper than primary cells, but their recombination pathways are often less stringent. Therefore, you should ideally achieve a high level of recombination efficiency before moving to primary lines.

In the end, efficiency of your CRISPR experiment is part plan, part luck. The interaction between the system components and Cas9 is still not well understood. Fortunately, there are a few ways you can increase your odds:

If you have done everything right but are still experiencing low efficiency, then it is time to experiment. You may have better luck using sense and anti-sense templates. Others have reported better efficiency with asymmetrical arms.5 Be prepared to design a few setups the efficiencies of overlapping designs can vary widely and be ready to experiment to find the best design for your experiments. For more information about CRISPR, check out this free CRISPR handbook.

How to Optimize Your Lentiviral ExperimentsMarch 7, 2017

There are several aspects to consider if you want to optimize your lentiviral experiments. Check out these helpful tips before you embark on the incessant optimization experiments. Here are three common factors that may be affecting your viral titers:

The 293 cell line was derived from embryonic kidney cells and is commonly used for lentivirus production. HEK 293 cells are sensitive to passage number and should be replaced regularly; cells must be healthy and actively dividing. HEK 293Ts, which contain the SV40 T antigen, are more resilient and can be used for six months or longer with no significant reduction in virus titer.

Clumpy cell cultures with lots of senescent cells will not produce good titers. It is worth doing a test transfection on your cells before you try using them for virus production. If your transfection efficiency is low, then there is no point continuing with virus production, you will need to setup a new cell stock.

Remember:

There are a number different commercial and non-commercial transfection reagents available. Chemical reagents such as calcium phosphate and polyethylenimine (PEI) work effectively and are very budget-friendly. For transfection with PEI or a commercial lipid-based reagent, your 293 cells should be 90-95% confluent at the time of transfection.

Remember:

A lentivirus expression typically contains a transfer plasmid and a packaging plasmid. Plasmids are recommended to be cultivated from bacterial strains such as Stbl3, which have reduced frequencies of homologous recombination. Plasmids containing a Gateway cassette with the ccdBgene will require a compatible ccdB viable strain. Make sure after plasmid purification that plasmid quality is high and of a reasonable concentration (over 100ng/L).

When considering packaging plasmids, make sure not to confuse the second and third generation variants. Second generation transfer plasmids require the presence of HIV-1 Tat protein. Third generation transfer plasmids have eliminated Tat from the packaging system, but are still backwards compatible with second generation transfer plasmids.

Transfer plasmids are the most important factor in virus production, and can result in transduction efficiency differences of 10-50x. The length of sequence between the long terminal repeats can directly influence viral titer, and particle yield decreases as sequence length increases. Including multiple promoters within the transfer plasmid can also result in promoter interference, where the promoters adversely affect expression of the others, resulting in lower viral titer.

Fluorescence microscopy and flow cytometry are two methods that can be used to measure transduction efficiency. Remember though that protein expression can influence fluorescence, and weakly expressed proteins can lead to underestimated viral titer. Therefore, promoter should be a key consideration if transduction is assessed using these methods.

Remember:

Researchers uncover novel fat metabolism pathwayFebruary 20, 2017

A new study in Nature Communications discovered a neuropeptide hormone, FLP-7, which is capable of stimulating fat metabolism. This fat metabolism pathway is the first to be discovered which can activate fat burning without affecting food intake or movement.

FLP-7 had previously been identified over 80 years ago as a muscle stimulant, but no links to fat metabolism was ever established. Flashing forward to 2017, scientists at the Scripps Institute identified FLP-7 during a genetic screen as a suppressor of fat loss in C. elegans roundworms. By fluorescently tagging the hormone, researchers were able to track FLP-7 secretions from the brain in response to elevated serotonin levels. This FLP-7 could then be tracked through the circulatory system to the gut, where it activates fat burning.

Modifying serotonin levels results in serious side effects, broadly impacting food intake, movement and reproduction. Amazingly, adjusting levels of FLP-7 does not result in any obvious changes, worms continue to function normally, while just burning more fat. Researchers hope their finding spur additional research into the weight loss effects of FLP-7 mammalian homologs.

Palamiuc et al. A tachykinin-like neuroendocrine signalling axis couples central serotonin action and nutrient sensing with peripheral lipid metabolism.Nature Communications, 2017; 8: 14237 DOI:10.1038/ncomms14237

How does sugar effect health and aging?February 6, 2016

A new study in Cell Reports has linked sugar intake to lifespan. This process occurs through a newly discovered pathway in which sugar permanently reprograms gene expression, maintaining an altered state even if your diet has improved.

Using fruit flies as a model organism, researchers compared life span of flies consuming a 5% and 40% sugar diet. Any flies raised on the 40% sugar diet averaged a 7% shorter life span. Researchers discovered that excess sugar promotes insulin-signaling pathways which lead to the inactivation of FOXO. FOXO is a transcription factor which alters the expression levels of chromatin modifiers. Crucially, the reprogramming of these transcription networks could not be reversed upon a switch to the lower sugar diet. The study improves our understanding of how changes in diet and gene expression can affect the speed of aging.

Dobson et al.Nutritional Programming of Lifespan by FOXO Inhibition on Sugar-Rich Diets.Cell Reports, 2017; 18 (2): 299 DOI:10.1016/j.celrep.2016.12.029

How does vitamin C fight cancer?January 30, 2016

Vitamin C’s efficacy in cancer prevention has been hotly debated. But, new research has shown that direct, intravenous delivery of vitamin C can more than double survival rates of pancreatic cancer. By avoiding the digestive tract, scientists have been able to increase vitamin C levels in the blood by 100-500 times. And at these extreme concentrations, vitamin C is able to selectively kill cancer cells.

As Vitamin C breaks down through oxidation hydrogen peroxide is generated. Hydrogen peroxide is capable of forming free radicals which can be damaging to DNA. Interestingly, researchers discovered that tumor cells are much less efficient at removing hydrogen peroxide. Tumor cells were found to be deficient in catalase activity, the primary means of detoxifying hydrogen peroxide. On average, tumor cells were able to only metabolize hydrogen peroxide at half the rate of normal cells. And the addition of vitamin C to these tumor cells resulted in ATP depletion, DNA lesions, and cell growth reduced by more than 50%. Clinical trials pairing both high-dosage, intravenous vitamin C and chemotherapy are now underway and in Phase 2 testing.

Doskey et al.Tumor cells have decreased ability to metabolize H2O2: Implications for pharmacological ascorbate in cancer therapy.Redox Biology, 2016; 10: 274 DOI:10.1016/j.redox.2016.10.010

Postdoc vs Industry? Comparing the ReturnsJanuary 23, 2016

A new study published in Nature Biotechnology has found that biomedical postdoctoral opportunities provide diminishing returns in the labor market. Upon graduating, many aspiring postdocs will hope to land a career in tenure track academia, but only 20% of scientists ever manage to attain such a position. The impact from such a decision can be staggeringly high.

Taking a postdoctoral position can cost up to three years worth of lost salary over the first 15 years of a scientist’s career. In 2013, the median starting salary for postdocs in academia was $44,724, compared to $73,662 for postdocs in industry. The academic experience accrued does not improve salary potential either, as scientists switching to industry average salaries equivalent to new, entry-level employees. Overall, academics will average $12,002 lower than though who leave the field.

But current graduates should stay informed of their options, and measure the chance of landing a tenure-track position against the potential financial ramifications.

Kahn and Ginther, 2017. The impact of postdoctoral training on early careers in biomedicine.Nature Biotechnology 2017; 35 (1): 90 DOI: 10.1038/nbt.3766

New mechanism for cancer metastasis discoveredJanuary 16, 2016

Cell biologists at Mount Sinai have identified a combination of changes to oncogenic and tumor suppressor genes which allow for early dissemination of cancer cells before a primary tumor forms. These cells first migrate before attaining additional mutations which lead to uncontrolled cell proliferation. But, a majority of the disseminated cancer cells will remain quiescent. And due to their non-proliferative nature, these cells form a reservoir resistant to chemotherapy and other conventional cancer treatments.

This early dissemination is a result of the activation of the p38 and HER2 pathways. Pathway activation leads to a cell type transition from epithelial to mesenchymal cells, which promotes cell migration. This process occurs normally in development during the formation of mammary and pancreatic ducts. But, the over-activation of both pathways during oncogenesis instead allows cancer cells to migrate into the bloodstream and metastasize instead.

Harper et al., 2016. Mechanism of early dissemination and metastasis in Her2 mammary cancer.Nature DOI: 10.1038/nature20609

Hosseimi et al., 2016. Early dissemination seeds metastasis in breast cancer.Nature DOI: 10.1038/nature20785

Mechanism behind Zika microcephaly revealedJanuary 9, 2016

Zika infection during fetal development has been associated with microcephaly and other birth defects. New analysis of Zika viral proteins has identified the mechanism by which the virus damages brain cells.

Cell biologists at Boston Children’s Hospital have identified the viral enzyme NS3 as the main culprit in Zika-associated neural degeneration. NS3 functions in the cleavage and processing of other Zika viral proteins. But, NS3 also is capable of interacting with and damaging centrioles, which are required for spindle assembly and cell proliferation. These findings are corroborated by genetic studies which have identified an association between centriole stability and microcephaly.

NS3 may prove to be an important drug target for against Zika-related illnesses moving forward. NS3 inhibitors commonly used to protect against dengue, a related virus, were shown to be successful in preventing NS3 binding to centrioles.

Saey, Tina.Cell biologists learn how Zika kills brain cells, devise schemes to stop it ScienceNews ScienceNews, 13 Dec 2016

How Did Mammary Glands EvolveJanuary 2, 2016

Researchers have recently discovered a new network of genes and enhancers responsible for coordinating the formation of mammary glands. Interestingly, this regulatory network functions by hijacking existing limb development processes.

Hox genes are a subset of homeotic genes which control embryonic development and patterning. Hox genes have been shown to regulate limb, head, thoracic, abdomen, and mammary gland formation.

To better understand how some of these body structures evolved, geneticists at the University of Geneva and the Swiss Federal Institute of Technology in Lusanne screened for Hox gene activating sequences in the genome. One of the enhancer sequences identified, MBRE, was found to be responsible for activating Hoxd9, a gene required for mammary gland development. Interestingly, MBRE is conserved only in placental and marsupial mammals, and missing in egg laying mammals, such as the platypus.

But MBRE regulatory network is found to function in all tissues, indicating that the network was present prior to mammary gland evolution. The researchers propose that Hoxd gene regulation in mammary glands evolved by co-opting existing regulatory networks in other body structures.

CRISPR Gene Editing Tested in Humans for the First TimeDecember 12, 2016

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DIY CRISPR Kit – The ODIN

There is currently ~1 week time till shipment.

Due to the overwhelming number of emails we will not respond to emails asking when your item will be shipped. Understand we are doing our best to get it to you.

Comes with an example experiment that teaches you many molecular biology and gene engineering techniques.

Want to really know what this whole CRISPR thing is about? Why it could revolutionize genetic engineering? This kit includes everything you need to make precision genome edits in bacteria at home including Cas9, tracrRNA, crRNA and Template DNA template for an example experiment.

Includes example experiment to make a genome mutation(K43T) to the rpsL gene changing the 43rd amino acid, a Lysine(K) to a Threonine(T) thereby allowing the bacteria to survive on Strep media which would normal prevent its growth.

Kit contains enough materials for around 5 experiments or more

Protocol For Experiment

You can find the plasmid DNA sequences

Cas9 Plasmid

gRNA Plasmid

Some items in this kit need to be stored in a fridge and a freezer upon you receiving them.

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DIY CRISPR Kit – The ODIN

A Crispr Conundrum: How Cells Fend Off Gene Editing – The …

Human cells resist gene editing by turning on defenses against cancer, ceasing reproduction and sometimes dying, two teams of scientists have found.

The findings, reported in the journal Nature Medicine, at first appeared to cast doubt on the viability of the most widely used form of gene editing, known as Crispr-Cas9 or simply Crispr, sending the stocks of some biotech companies into decline on Monday.

Crispr Therapeutics fell by 13 percent shortly after the scientists announcement. Intellia Therapeutics dipped, too, as did Editas Medicine. All three are developing medical treatments based on Crispr.

But the scientists who published the research say that Crispr remains a promising technology, if a bit more difficult than had been known.

The reactions have been exaggerated, said Jussi Taipale, a biochemist at the University of Cambridge and an author of one of two papers published Monday. The findings underscore the need for more research into the safety of Crispr, he said, but they dont spell its doom.

This is not something that should stop research on Crispr therapies, he said. I think its almost the other way we should put more effort into such things.

Crispr has stirred strong feelings ever since it came to light as a gene-editing technology five years ago. Already, its a mainstay in the scientific tool kit.

The possibilities have led to speculations about altering the human race and bringing extinct species back to life. Crisprs pioneers have already won a slew of prizes, and titanic battles over patent rights to the technology have begun.

To edit genes with Crispr, scientists craft molecules that enter the nucleus of a cell. They zero in on a particular stretch of DNA and slice it out.

The cell then repairs the two loose ends. If scientists add another piece of DNA, the cell may stitch it into the place where the excised gene once sat.

Recently, Dr. Taipale and his colleagues set out to study cancer. They used Crispr to cut out genes from cancer cells to see which were essential to cancers aggressive growth.

For comparison, they also tried to remove genes from ordinary cells in this case, a line of cells that originally came from a human retina. But while it was easy to cut genes from the cancer cells, the scientists did not succeed with the retinal cells.

Such failure isnt unusual in the world of gene editing. But Dr. Taipale and his colleagues decided to spend some time to figure out why exactly they were failing.

They soon discovered that one gene, p53, was largely responsible for preventing Crispr from working.

p53 normally protects against cancer by preventing mutations from accumulating in cellular DNA. Mutations may arise when a cell tries to fix a break in its DNA strand. The process isnt perfect, and the repair may be faulty, resulting in a mutation.

When cells sense that the strand has broken, the p53 gene may swing into action. It can stop a cell from making a new copy of its genes. Then the cell may simply stop multiplying, or it may die. This helps protect the body against cancer.

If a cell gets a mutation in the p53 gene itself, however, the cell loses the ability to police itself for faulty DNA. Its no coincidence that many cancer cells carry disabled p53 genes.

Dr. Taipale and his colleagues engineered retinal cells to stop using p53 genes. Just as they had predicted, Crispr now worked much more effectively in these cells.

A team of scientists at the Novartis Institutes for Biomedical Research in Cambridge, Mass., got similar results with a different kind of cells, detailed in a paper also published Monday.

They set out to develop new versions of Crispr to edit the DNA in stem cells. They planned to turn the stem cells into neurons, enabling them to study brain diseases in Petri dishes.

Someday, they hope, it may become possible to use Crispr to create cell lines that can be implanted in the body to treat diseases.

When the Novartis team turned Crispr on stem cells, however, most of them died. The scientists found signs that Crispr had caused p53 to switch on, so they shut down the p53 gene in the stem cells.

Now many of the stem cells survived having their DNA edited.

The authors of both studies say their results raise some concerns about using Crispr to treat human disease.

For one thing, the anticancer defenses in human cells could make Crispr less efficient than researchers may have hoped.

One way to overcome this hurdle might be to put a temporary brake on p53. But then extra mutations may sneak into our DNA, perhaps leading to cancer.

Another concern: Sometimes cells spontaneously acquire a mutation that disables the p53 gene. If scientists use Crispr on a mix of cells, the ones with disabled p53 cells are more likely to be successfully edited.

But without p53, these edited cells would also be more prone to gaining dangerous mutations.

One way to eliminate this risk might be to screen engineered cells for mutant p53 genes. But Steven A. McCarroll, a geneticist at Harvard University, warned that Crispr might select for other risky mutations.

These are important papers, since they remind everyone that genome editing isnt magic, said Jacob E. Corn, scientific director of the Innovative Genomics Institute in Berkeley, Calif.

Crispr doesnt simply rewrite DNA like a word processing program, Dr. Corn said. Instead, it breaks DNA and coaxes cells to put it back together. And some cells may not tolerate such changes.

While Dr. Corn said that rigorous tests for safety were essential, he doubted that the new studies pointed to a cancer risk from Crispr.

The particular kinds of cells that were studied in the two new papers may be unusually sensitive to gene editing. Dr. Corn said he and his colleagues have not found similar problems in their own research on bone marrow cells.

We have all been looking for the possibility of cancer, he said. I dont think that this is a warning for therapies.

We should definitely be cautious, said George Church, a geneticist at Harvard and a founding scientific adviser at Editas.

He suspected that p53s behavior would not translate into any real risk of cancer, but its a valid concern.

And those concerns may be moot in a few years. The problem with Crispr is that it breaks DNA strands. But Dr. Church and other researchers are now investigating ways of editing DNA without breaking it.

Were going to have a whole new generation of molecules that have nothing to do with Crispr, he said. The stock market isnt a reflection of the future.

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A Crispr Conundrum: How Cells Fend Off Gene Editing – The …

GeneHero CRISPR Products and Services | Genecopoeia

GeneCopoeia’s GeneHero CRISPR-Cas9 products and services provide a complete, powerful solution to your genome editing needs. Products and services include:

CRISPR Plasmids. Transfect cells with our CRISPR plasmids with Cas9 and sgRNA for human, mouse, and rat. Search our database of more than 45,000 human, mouse, and rat genes for genome editing using CRISPR.

CRISPR Lentivirus.Genome integration of CRISPR elements using lentivirus. Cas9 and/or sgRNA packed in purified lentiviral particles at 108 TU/ml, ready to infect all cell types.

CRISPR AAV.Episomal expression of CRISPR components with adeno-associated viralparticles carrying Cas9 and/or sgRNA, excellent for tissue and animal transduction.

Cas9 Stable Cell Lines.Premade Cas9-expressing stable cell lines are great for sgRNA library screening and other high-throughput CRISPR-Cas9 applications.

The clustered, regularly interspaced, short palindromic repeats (CRISPR) system is bacterial immunity mechanism for defense against invading viruses and transposons. This system has been adapted for highly efficient genome editing in many organisms. Compared with earlier genome editing technologies such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), CRISPR-Casmediated gene targeting has similar or greater efficiency. Genome editing has been used for numerous applications, as shown in Table 1.

Table 1. Applications for CRISPR-mediated genome editing.

In the type II CRISPR systems, the complex of a CRISPR RNA (crRNA) annealed to a trans-activating crRNA (tracrRNA) guides the Cas9 endonuclease to a specific genomic sequence, thereby generating double-strand breaks (DSBs) in target DNA. This system has been simplified by fusing crRNA and tracrRNA sequences to produce a synthetic, chimeric single-guided RNA (sgRNA). The sgRNA contains within it a 20 nucleotide DNA recognition sequence (Figure 1).

Figure 1. Mechanism of CRISPR-Cas9-sgRNA target recognition and cleavage.

When the Cas9-sgRNA complex encounters this target sequence in the genome followed by a 3 nucleotide NGG PAM (protospacer adjacent sequence) site, the complex binds to the DNA strand complementary to the target site. Next, the Cas9 nuclease creates a site-specific double-strand break (DSB) 3-4 nucleotides 5′ to the PAM. DSBs are repaired by either non-homologous end joining (NHEJ), which is error-prone, and can lead to frameshift mutations, or by homologous recombination (HR) in the presence of a repair template (Figure 2).

Figure 2.CRISPR-Cas9-based gene engineering. Left. DSBs created by sgRNA-guided Cas9-mediated cleavage are repaired by NHEJ. Right. DSBs created by sgRNA-guided Cas9 nuclease are repaired homologous recombination between sequences flanking the DSB site, thereby causing “knock in” of sequences on a donor DNA.

While the CRISPR system provides a highly efficient means for carrying out genome editing applications, it is prone to causing off-target indel mutations. Off-targeting is caused by the ability of the Cas9- sgRNA complex to bind to chromosomal DNA targets with one or more mismatches, or non-Watson-Crick complementary. The propensity of CRISPR for off-target modification is a significant concern for some researchers who want to avoid results that are potentially confounded by off-target modification, as well as for those who might be interested in developing CRISPR for gene therapy applications.

Several strategies have been employed to mitigate CRISPR’s propensity for off-target genome modification. One such strategy is to use double nickases to create DSBs. The Cas9 D10A mutant is able to cleave only one DNA strand, thereby creating a “nick”. When two sgRNAs that bind on opposite strands flanking the target are introduced, two Cas9 D10A nickase molecules together create a staggered-cut DSB, which is then repaired by either NHEJ or HR (Figure 3). The double nickase strategy has been shown to greatly reduce the frequency of off-target modification. However, double nickases are limited in utility by design constraints; the sgRNAs must be on opposite strands, in opposite orientation to one another, and display optimal activity when spaced from 3-20 nucleotides apart. In addition, the cleavage activity of double nickases tends to be lower than that of standard Cas9-sgRNA. Further, nickases can still cause some degree of off-target indel formation.

Figure 3. General scheme of Cas9 double-nickase strategy. From Ran, et al. (2013). Two additional strategies, the use of truncated (17-18 nucleotide) sgRNAs, as well as a Cas9-FokI fusion, also dramatically reduce CRISPR-mediated off-target genome modification. However, these methods suffer from even further reductions in on-target activity and/or more severe design constraints compared with the double nickase approach.

Recently, two groups demonstrated that engineering Cas9 variants carrying 3-4 amino acid changes virtually eliminates CRISPR off-target genome modification. These variants still retain high on-target activity, without the design constraints of previous approaches, providing a promising alternative for high-fidelity CRISPR-mediated genome editing.

Watch recorded webinar / Download slides Title: Genome Editing: How Do I Use CRISPR? Presented Wednesday, February 22, 2017

Genome Editing-the ability to make specific changes at targeted genomic sites-is fundamentally important to researchers in biology and medicine. CRISPR is a very widely-used method for modifying specific genome sites, and can be used for many applications, including gene knock out, transgene knock in, gene tagging, and correction of genetic defects. However, researchers are often unaware of some of the work required to identify their desired modification in their cell lines. In this webinar, we discuss what you need to do for CRISPR genome editing after you have obtained your reagents from GeneCopoeia, the so-called Downstream work.

Watch recorded webinar / Download slides Title: GeneCopoeia CRISPR Genome Editing Technology Presented Wednesday, January 25, 2017

The ability to make specific changes at targeted genomic sites in complex organisms is fundamentally important to researchers in biology and medicine. Researchers have developed and refined chimeric DNA endonucleases, such as CRISPR-Cas9, to stimulate double strand breaks at defined genomic loci, allowing the ability to insert, delete, and replace genetic information at will. These tools can also be used without nucleases to induce or repress gene transcription. In this webinar, we discuss CRISPR and other genome editing technologies and the applications they make possible, and provide information on GeneCopoeia’s powerful suite of genome editing products and services.

Watch recorded webinar / Download slides Title: Applications For CRISPR-Cas9 Stable Cell Lines Presented Wednesday, March 22, 2017

The CRISPR-Cas9 system has become greatly popular for genome editing in recent years, due to its ease-of-design, efficiency, specificity, and relatively low cost. In mammalian cell culture systems, most genome editing is achieved using transient transfection or lentiviral transduction, which works well for routine, low-throughput applications. However, for other applications, it would be beneficial to have a system in which one component, namely the CRISPR-Cas9 nuclease, was stably integrated into the genome. In this webinar, we introduce GeneCopoeias suite of Cas9 stable cell lines, and discuss the great utility that these cell lines provide for genome editing applications.

Watch recorded webinar / Download slides Title: Safe Harbor Transgenesis in Human & Mouse Genome Editing Presented Wednesday, April 19, 2017

Insertion of transgenes in mammalian chromosomes is an important approach for biomedical research and targeted gene therapy. Traditional lentiviral-mediated transgenesis is effective and straightforward, but its random integration can often be unstable and harm cells. “Safe Harbor” sites in human and mouse chromosomes have been employed recently as an alternative to random, viral-mediated integration because they support consistent, stable expression, and are not known to hamper cell fitness or growth. In this webinar, we will discuss the merits of Safe harbor transgenesis approaches, and how GeneCopoeia’s CRISPR tools for Safe Harbor knock-in can greatly benefit your research.

Watch recorded webinar / Download slides Title: GeneCopoeia CRISPR sgRNA Libraries For Functional Genomics Presented Wednesday, April 29, 2015

Biomedical researchers are enjoying a Renaissance in functional genomics, which aims to use a wealth of DNA sequence informationmost notably, the complete sequence of the human genometo determine the natural roles of the genes encoded by the genome. As a result, biochemical networks and pathways will be better understood, with the hope of leading to improved disease treatments. Researchers are turning increasingly to CRISPR (clustered, regularly interspaced, short palindromic repeats) for functional genomics studies. Several groups recently adapted CRISPR for high-throughput knockout applications, by developing large-scale CRISPR sgRNA libraries. GeneCopoeia recently launched a number of smaller, pathway- and gene group-focused CRISPR sgRNA libraries, which offer several key advantages over the whole-genome libraries. In this 40 minute webinar, we discuss the merits and applications for CRISPR sgRNA libraries, how to use CRISPR sgRNA libraries, the advantages of using small, pathway- and gene group-focused libraries, and how GeneCopoeia can help you with your high-throughput CRISPR knockout studies.

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Answer:If you are doing simple gene knockouts in humans or mice, you can order CRISPR sgRNAs on our website. All you need to do is go to the , search for your gene, and then choose the appropriate clones that will work for your system. These CRISPR sgRNAs are designed by default to knock out all possible known and predicted transcript variants of your gene, and are targeted early in the coding regions. You can also order donor clones for these knockouts from the search results page. If you are doing a different application, such as introducing a point mutation, then you will need to and, after determining what you need, we will send you a custom quote.

Answer:For sgRNA clones (including both all-in-one Cas9/sgRNA clones and sgRNA-only clones, the default delivery format is bacterial stock. You have the option of ordering purified DNA for these clones for an additional charge. For HDR donor clones, the default delivery format is purified DNA.

Answer:The turnaround time for sgRNA clones (including both all-in-one Cas9/sgRNA clones and sgRNA-only clones) is 2-3 weeks. The turnaround time for HDR donor clones depends greatly on the nature of the modification that the clone is being used for. For HDR donor clones used for simple knockout, the turnaround time is 2-4 weeks. Other HDR donor clones, such as those used for fusion tagging or mutagenesis, can take 6-8 weeks, but can also take longer.

Answer:Yes. We sequence the inserts of each CRISPR sgRNA clone, and provide you with datasheets that show the full sequence of each clone (including HDR donor clones), a map, restriction enzyme digestions sites, and suggested sequencing primers. To obtain these datasheets, you just need to visit our on our website. You will need an account on our website, your catalog number(s), and your sales order number.

Answer:In the presence of drug, the only way for cells to survive is to integrate the plasmid into the chromosome, so it is possible to get drug-resistant clones that were only transfected with the donor plasmid. However, such integration is random. CRISPR increases donor targeting frequency by several orders of magnitude.

Answer:Our genome editing products can be used for virtually all species. Our standard plasmids for CRISPR are designed for work in mammalian cells. In addition, these plasmids can be used as templates for T7 promoter-driven in vitro transcription, for introduction into mice, zebrafish, Drosophila, and many other model organisms. Further, we can generate custom constructs that can be used in a wide variety of organisms.

Answer:Yes. The donor must be present when the DSB is formed in order to be used as a repair template. Otherwise, the cell must use non-homologous end joining (NHEJ) in order to repair the DSB, because unrepaired DSBs are lethal.

Answer:Our CRISPR plasmids typically do not integrate into the host genome in transfection experiments. However, after clonal selection for edited cells, we recommend screening clones for those which have lost the nuclease plasmids. This can be done by testing clones to see if they have become sensitive to the antibiotic of the resistance gene on the plasmid, or if they no longer express the plasmid’s fluorescent marker (where applicable). Our lentiviral clones are expected to integrate randomly into chromosomes.

1. If you are making an insertion or deletion, the easiest way to screen your cells is by PCR using primers flanking the modified site, provided that the insertion or deletion is large enough to detect by standard agarose gel electrophoresis.

2. For very small insertions or deletions, you can screen your clones using GeneCopoeia’s IndelCheck T7 endonuclease I assay, which is a method that detects mutations by cleaving double stranded DNA containing a mismatch. You can also screen using Sanger sequencing of PCR products.

3. If you are introducing a point mutation, then you can use either real-time PCR or Sanger sequencing to detect the modification.

4. If the modification you are introducing creates or destroys a restriction enzyme site, then enzyme cleavage of PCR products can be used to distinguish between modified and unmodified alleles.

5. Finally, either Sanger sequencing of PCR products or Next Generation sequencing of whole genomes can be used to screen for modifications. Regardless of which screening method you choose, it is also important that you are able to determine whether only a portion or all of the alleles have been modified.

In order to reduce the amount of time and effort required to identify edited clones, GeneCopoeia recommends our donor plasmid design and construction service. We will construct a donor plasmid that contains a defined modification, flanked by a selectable marker such as puromycin resistance, and homologous arms from your target region. The donor may or may not also include a fluorescent reporter such as GFP. The markers can be flanked by loxP sites, to permit Cre-mediated removal, if desired. Use of a GeneCopoeia-designed donor plasmid allows you to select for edited clones and reduces the number of clones required for screening. You can also purchase our donor cloning vectors for do-it-yourself donor clone construction.

Answer:Yes. Even though frameshifts are not possible with miRNAs and other noncoding RNAs, an indel occurring in a critical region, such as the mature sequence of a miRNA, should be enough to abolish its function.

Answer:The vector backbones of our CRISPR sgRNAs are designed to not replicate in the host. These plasmids, which are transiently transfected, will typically be lost after several rounds of cell division and will not further affect the host cell. After transfection, cells are plated at low density to promote the formation of single colonies. These colonies should be screened to ensure that they have lost the plasmid(s). This can be done by testing clones to see if they have become sensitive to the antibiotic of the resistance gene on the plasmid, or if they no longer express the plasmid’s fluorescent marker (where applicable). However, even if the TALEN or CRISPR plasmid integrates, it can no longer cut the site after it is edited, because NHEJ destroys the TALEN or sgRNA recognition site. To be completely assured that the transfection is transient, we recommend delivering RNA instead of plasmid DNA. If you are using HDR, we recommend engineering synonymous mutations into the donor to destroy the TALEN or sgRNA recognition site.

Answer:Yes. CRISPR has been shown to be able to disrupt multiple copies at once. The efficiency varies depending on different factors, such as cell type, transfection efficiency and TALEN/CRISPR activity.

Answer:Yes. We have the reagents for the Cas9 D10A nickase, and have successfully tested our double nickase designs. However, in order to create mutagenic DSBs, the nickase requires the correct targeting of two appropriately-spaced sgRNAs on opposite strands, flanking the break site. Because proper sgRNA targeting requires the presence of the N-G-G PAM site immediately following the recognition site, it might not always be possible to use the nickase for DSB formation. There are also high-fidelity variants of Cas9 nuclease that edit genes with greater specificity than wild type Cas9, but sometimes with reduced efficacy and with increased design constraints. However, since these high fidelity variants use only one sgRNA, they are easier to work with than Cas9 niclases.

Answer:Yes. To create a DSB, the nickase requires the correct targeting of two appropriately-spaced sgRNAs on opposite strands, flanking the break site. This is sufficient to stimulate HDR between the target site and the donor. While this method has the advantage of potentially fewer off-target NHEJ-mediated mutations, since single strand nicks are repaired with higher fidelity than DSBs, it is not without limitations. Proper sgRNA targeting requires the presence of the N-G-G PAM site immediately following the recognition site. Therefore, it might not always be possible to use the nickase for HDR.

Answer:We only sell plasmids containing our custom-designed CRISPR sgRNAs. If you need a negative control, we also sell a CRISPR plasmid containing a scrambled sgRNA.

Answer:Yes.

Answer:Yes. There is a double mutant of the Cas9 nuclease that completely abolishes nuclease activity. This mutant can be fused to a transcriptional modulator such as VP64 and targeted to specific genes. You can also use the catalytically dead Cas9 with properly-designed sgRNAs to repress, or interfere with, gene expression.

Answer:Yes. We have both non-viral and lentiviral formats. We also have , in which we can provide you with lentiviral particles expressing both Cas9 and sgRNAs.

Answer:Unfortunately, no. Lentiviruses enter cells as RNA, but HDR donors must enter the cells as DNA at the same time as Cas9 and the sgRNAs.

Answer:Lentiviral particles, transfection-ready DNA, and bacterial stock.

Answer:Yes. The lentiviral plasmids are “dual-use”, so that they can either be packaged into lentiviral particles or transfected into cells by standard transfection methods.

Answer:Our sgRNA representation does not need to be validated by Next Generation Sequencing. Each library is small compared with the genome-wide libraries, and each sgRNA clone is constructed individually, cultured in E. coli individually, then pooled as E. coli in approximately equal amounts. From those pools we prepare DNA and then, if necessary, lentiviral particles.

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GeneHero CRISPR Products and Services | Genecopoeia

CRISPR Background CRISPR Update

Targeted gene editing began with the discovery of zinc finger proteins in the 1980s and continued to improve through the 1990s and early 2000s with the discovery of Transcription activator-like effector nucleases, or TALENS (1, 2). Both of these techniques rely on complex protein structures being engineered to target specific DNA sequences and containing a fused nuclease that nicks a single strand of the DNA duplex. In order to induce the double stranded break (DSB) needed for non-homologous end joining (NHEJ) or homology directed repair (HDR) two zinc finger or TALEN proteins are needed, each targeting one strand of the DNA duplex. While these techniques are reliable, the challenges in designing the protein structures needed to target specific DNA sequences limited their widespread adoption. In 2012 the CRISPR/Cas system was found to target and cut specific DNA sequences using only a nuclease and RNAs to target specific DNA sequences (3). The ease with which this system allows for targeting any gene has set off a new era in targeted gene editing.

Clustered Regular Interspaced Palindromic Repeats, or CRISPRs, were originally identified in the late 1980s in bacteria as short segments of repeating DNA separated by unique spacer sequences however their significance was originally, not known (4). It was not until the early 2000s that the term CRISPR was coined and specific genes, named CRISPR-Associated genes, or Cas genes, were identified (5). Throughout the next decade it was found that the unique spacer sequences were homologous to phage DNA and that certain Cas proteins (i.e. Cas9) used transcribed CRISPR RNA to target and cleave phage DNA, thus acting as an adaptive immune system for bacteria (3, 610). The CRISPR system is composed of two RNA components, crRNA and tracrRNA. Both are transcribed and are required for Cas9 cleavage activity (7). The crRNA is the RNA moiety that targets a specific gene sequence; it contains the transcribed unique spacer RNA as well as a palindromic repeat. The tracrRNA contains a palindromic repeat (the complementary sequence to the crRNA) and a region that can bind to Cas9. Upon duplexing of the crRNA and tracrRNA, this RNA complex can join with Cas9 to target DNA complementary to the unique spacer region of the crRNA (3). Once the crRNA forms a duplex with DNA and the PAM sequence is engaged, Cas9 will cut both strands of the DNA resulting in a double stranded break (DSB), thereby inducing the host DNA repair mechanisms.

After cleavage, DNA can by repaired one of two ways. The simplest, most efficient repair mechanism is referred to as Non-Homologous End-Joining (NHEJ) repair and is the result of enzymes adding and/or removing DNA bases at random to repair the break. This process can result in mutations, by either introducing a premature stop codon or by causing a frameshift mutation. Either one of these mutations ultimately results in a non-functional gene product. NHEJ is routinely used when researchers want to knockout a specific gene. Less efficient than NHEJ is Homology Directed Repair (HDR). HDR is used to insert/knockout genes or to make a specific change at a DSB. In addition to needing the CRISPR/Cas9 machinery, HDR requires a sequence of DNA whose ends are homologous to the ends of the DSB. After inducing a DSB, the cell inserts the new sequence through homologous recombination. To induce specific mutations in cells lines, addition of a donor DNA is needed.

In 2012 Jennifer Doundas group at University of California-Berkley characterized the activity of Cas9 and found that the two RNA component of Cas9 could be modified into a single strand of RNA. This new RNA fragment was coined the guide RNA (gRNA), also known as a single guide RNA. The gRNA is composed of a truncated tracrRNA sequence coined the scaffold sequence fused to a ~20 nucleotide user defined spacer or targeting sequence (3). This system can theoretically be used to target any sequence in a genome provided it meets two conditions. First, the sequence must be unique when compared to the rest of the genome and second, the target sequence has to be immediately followed by the Protospacer Adjacent Motif (PAM). The PAM is a 3-5 nucleotide sequence that is required for Cas cleavage activity. Cas9 has a three nucleotide PAM NGG while other Cas proteins have been identified with different PAM sequences (11). Additionally, protein engineering has been used to create Cas9 variants with different PAM sequences thus expanding the number of genomic targets possible.

Identification of CRISPR mutations depends on which repair mechanism is employed. When large genes are inserted by HDR, PCR amplification of the transgene can easily identify which lines are positive for the desired event. When HDR is used to repair small sections of DNA that do not result in large insertions, sequencing or heteroduplex cleavage are used to identify the changes. A DSB repaired via NHEJ can be detected using a heteroduplexing and endonuclease assay such as T7EI or Surveyor. Upon heteroduplexing of the mutated sequence with a wild-typesequence, T7EI or Surveyor can cleave at the mismatched DNA bases. Successfully modified sequences are then identified by comparing the fragment sizes produced by the assay with the theoretical fragment size of the CRISPR targeted sequence. The ease at which CRISPR/Cas systems can be programed to target virtually any gene in any genome potentially allows for widespread adoption in a number of industries and applications. . Right now, CRISPR is being used to understand how different genes impact human disease through the use of several model animal systems. It is also being used to engineer the next generation of production crops and animals. In the more immediate future CRISPR gene editing may be used to potentially fight widespread zoonotic diseases such as malaria. The applications are endless. While no one can be certain how far reaching the impact CRISPR technology will be, it has undoubtedly revolutionized molecular biology.

Continued here:
CRISPR Background CRISPR Update

Antibodies Part 1: CRISPR – Radiolab

Hidden inside some of the worlds smallest organisms is one of the most powerful tools scientists have ever stumbled across. It’s a defense system that has existed in bacteria for millions of years and it may some day let us change the course of human evolution.

Out drinking with a few biologists, Jad finds out about something called CRISPR. No, its not a robot or the latest dating app, its a method for genetic manipulation that is rewriting the way we change DNA. Scientists say theyll someday be able to use CRISPR to fight cancer and maybe even bring animals back from the dead. Or, pretty much do whatever you want. Jad and Robert delve into how CRISPR does what it does, and consider whether we should be worried about a future full of flying pigs, or thesimple fact that scientists have now used CRISPR to tweak the genes of human embryos.

As of February 24th, 2017 we’ve updated this story.

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Antibodies Part 1: CRISPR – Radiolab

Crispr gene editing ready for testing in humans – ft.com

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Ever since scientists began decoding the human genome in 1990, doctors have dreamt of a new era of medicine where illness could be treated or even cured by fxing flaws in a persons DNA. Rather than using medicine to fight disease, they would be able to hack biology to combat sickness at its source.

The dream started to become a reality in 2013, when researchers demonstrated how a gene editing technique, known as Crispr-Cas9, could be used to edit living human cells, raising the possibility that a persons DNA could be altered much as text is changed by a word-processor.

Now, two biotech companies say they plan to start testing the technology in humans as early as this year.

Crispr Therapeutics has already applied for permission from European regulators to test its most advanced product, code-named CTX001, in patients suffering from beta-thalassaemia, an inherited blood disease where the body does not produce enough healthy red blood cells. Patients with the most severe form of the illness would die without frequent transfusions.

The Switzerland-based company says it also plans to seek a greenlight from the US Food and Drug Administration this year so it can trial CTX001 in people with sickle cell disease, another inherited blood disorder.

Editas Medicine, Crisprs US-based rival, says it plans to apply for permission from the FDA in the middle of the year so it can test one of its one of its own Crispr gene-editing products in patients with a rare form of congenital blindness that causes severe vision loss at birth. If the FDA agrees, it should be able to commence trials within 30 days of the application.

If those trials are successful, Crispr, Editas and a third company, Intellia Therapeutics, say they plan to study the technique in humans with a range of diseases including cancer, cystic fibrosis, haemophilia and Duchenne muscular dystrophy.

In China, where regulators have taken a more lenient approach to human trials, several studies are already under way, although they have yet to produce any conclusive data.

Crispr-Cas9 is best thought of as two technologies that make gene editing possible: Cas9 acts as a pair of molecular scissors that can snip strands of DNA, removing faulty genetic material and creating space for functioning genes to be inserted. Crispr is a kind of genetic GPS that guides those scissors to the precise location.

Katrine Bosley, chief executive of Editas, says the field of gene editing is moving at lightning speed, but that the technique will at first be limited to illnesses where there are not other good options.

That is because, as with any new technology, scientists and regulators are not fully aware of the safety risks involved. We want it to be as safe as it can, but of course there is this newness, says Ms Bosley.

Francisco Mojica at the University of Alicante, Spain becomes the first researcher to discover Crispr sequences

Alexander Bolotin at the French National Institute for Agricultural Research observes Cas9 genes in the bacteria Streptococcus thermophilus

Scientists at Danone study how Crispr techniques can help Streptococcus thermophilus, widely used in commercial yoghurt making, ward off viral attacks

Biochemists Jennifer Doudna and Emmanuelle Charpentiere show that Crispr can be used to edit DNA in test tubes

Feng Zhang of the Broad Institute reports using Crispr to edit DNA in human cells, opening the door for the tool to be used in medicine

Crispr is used to edit the genomes of everything from flies to mice

British scientists use Talen gene editing to treat a childs leukaemia

Still, Ms Bosley points out that of the more than 6,000 genetic disorders, which are the most obvious candidates for gene editing, roughly 95 per cent are untreatable. This provides plenty of areas for companies like hers to explore.

Although Crispr-Cas9 has not yet been trialled in humans in Europe or the US, the technology has already benefited medical research greatly by speeding up laboratory work. It used to take scientists several years to create a genetically modified mouse for their experiments, but with Crispr-Cas9 these transgenic mice can be produced in a few weeks.

Cellectis, a French biotech group, has used an older gene-editing technique known as Talen, to create a pioneering blood cancer treatment known as chimeric antigen receptor therapy or Car-T, which is currently being tested in humans.

Car-T products are already on the market, but rely on an expensive and laborious process that involves extracting a persons white blood cells, transporting them by aeroplane to a lab where they are re-engineered to attack cancer, before returning them and inserting them into the patient.

Cellectis hopes its approach of using gene editing to alter the cells will cut out this lengthy re-engineering process.

Some proponents of Crispr-Cas9 dismiss the Talen technique as old, slow and expensive, but Andr Choulika, Cellectis chief executive, disagrees.

We asked readers, researchers and FT journalists to submit ideas with the potential to change the world. A panel of judges selected the 50 ideas worth looking at in more detail. This fourth tranche of 30 ideas (listed below) is about the latest advances in healthcare. The fifth and final chapter, looking at Earth and the universe, will be published on March 29, 2018.

Were not talking about iPhones here, he says. Maybe [Crispr] is a new technology, its easy to design and its cheap, but who cares? This is not what the patient needs. The patient needs a super-active, super-precise product.

Amid the excitement, the nascent field of gene editing has been hampered by several setbacks. Editas had hoped to start human trials earlier, but was forced to move the date back after it encountered manufacturing delays. Crispr has lost several key executives in recent months, while Cellectis had to suspend its first trial briefly last year after a patient died.

Meanwhile, a bitter patent dispute over which academic institution discovered Crispr-Cas9, and therefore which biotech company has the rights to the patents, has cast a pall over gene editing.

The field is in its infancy and progress in any new area of science is never smooth. If gene editing lives up to its promise, it could one day save or dramatically change the lives of tens of millions of patients with hitherto untreatable diseases.

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Crispr gene editing ready for testing in humans – ft.com

CRISPR – YouTube

Designer babies, the end of diseases, genetically modified humans that never age. Outrageous things that used to be science fiction are suddenly becoming reality. The only thing we know for sure is that things will change irreversibly.

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SOURCES AND FURTHER READING:

The best book we read about the topic: GMO Sapiens

https://goo.gl/NxFmk8

(affiliate link, we get a cut if buy the book!)

Good Overview by Wired:http://bit.ly/1DuM4zq

timeline of computer development:http://bit.ly/1VtiJ0N

Selective breeding: http://bit.ly/29GaPVS

DNA:http://bit.ly/1rQs8Yk

Radiation research:http://bit.ly/2ad6wT1

inserting DNA snippets into organisms:http://bit.ly/2apyqbj

First genetically modified animal:http://bit.ly/2abkfYO

First GM patent:http://bit.ly/2a5cCox

chemicals produced by GMOs:http://bit.ly/29UvTbhhttp://bit.ly/2abeHwUhttp://bit.ly/2a86sBy

Flavr Savr Tomato:http://bit.ly/29YPVwN

First Human Engineering:http://bit.ly/29ZTfsf

glowing fish:http://bit.ly/29UwuJU

CRISPR:http://go.nature.com/24Nhykm

HIV cut from cells and rats with CRISPR:http://go.nature.com/1RwR1xIhttp://ti.me/1TlADSi

first human CRISPR trials fighting cancer:http://go.nature.com/28PW40r

first human CRISPR trial approved by Chinese for August 2016:http://go.nature.com/29RYNnK

genetic diseases:http://go.nature.com/2a8f7ny

pregnancies with Down Syndrome terminated:http://bit.ly/2acVyvg( 1999 European study)

CRISPR and aging:http://bit.ly/2a3NYAVhttp://bit.ly/SuomTyhttp://go.nature.com/29WpDj1http://ti.me/1R7Vus9

Help us caption & translate this video!

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CRISPR – YouTube

Researchers advance CRISPR-based tool for diagnosing disease …

The team that first unveiled the rapid, inexpensive, highly sensitive CRISPR-based diagnostic tool called SHERLOCK has greatly enhanced the tools power, and has developed a miniature paper test that allows results to be seen with the naked eye without the need for expensive equipment.

The SHERLOCK team developed a simple paper strip to display test results for a single genetic signature, borrowing from the visual cues common in pregnancy tests. After dipping the paper strip into a processed sample, a line appears, indicating whether the target molecule was detected or not.

This new feature helps pave the way for field use, such as during an outbreak. The team has also increased the sensitivity of SHERLOCK and added the capacity to accurately quantify the amount of target in a sample and test for multiple targets at once. All together, these advancements accelerate SHERLOCKs ability to quickly and precisely detect genetic signatures including pathogens and tumor DNA in samples.

Described today in Science, the innovations build on the teams earlier version of SHERLOCK (shorthand for Specific High Sensitivity Reporter unLOCKing) and add to a growing field of research that harnesses CRISPR systems for uses beyond gene editing. The work, led by researchers from the Broad Institute of MIT and Harvard and from MIT, has the potential for a transformative effect on research and global public health.

SHERLOCK provides an inexpensive, easy-to-use, and sensitive diagnostic method for detecting nucleic acid material and that can mean a virus, tumor DNA, and many other targets, said senior author Feng Zhang, a core institute member of the Broad Institute, an investigator at the McGovern Institute, and the James and Patricia Poitras 63 Professor in Neuroscience and associate professor in the departments of Brain and Cognitive Sciences and Biological Engineering at MIT. The SHERLOCK improvements now give us even more diagnostic information and put us closer to a tool that can be deployed in real-world applications.

The researchers previously showcased SHERLOCKs utility for a range of applications. In the new study, the team uses SHERLOCK to detect cell-free tumor DNA in blood samples from lung cancer patients and to detect synthetic Zika and Dengue virus simultaneously, in addition to other demonstrations.

Clear results on a paper strip

The new paper readout for SHERLOCK lets you see whether your target was present in the sample, without instrumentation, said co-first author Jonathan Gootenberg, a Harvard graduate student in Zhangs lab as well as the lab of Broad core institute member Aviv Regev. This moves us much closer to a field-ready diagnostic.

The team envisions a wide range of uses for SHERLOCK, thanks to its versatility in nucleic acid target detection. The technology demonstrates potential for many health care applications, including diagnosing infections in patients and detecting mutations that confer drug resistance or cause cancer, but it can also be used for industrial and agricultural applications where monitoring steps along the supply chain can reduce waste and improve safety, added Zhang.

At the core of SHERLOCKs success is a CRISPR-associated protein called Cas13, which can be programmed to bind to a specific piece of RNA. Cas13s target can be any genetic sequence, including viral genomes, genes that confer antibiotic resistance in bacteria, or mutations that cause cancer. In certain circumstances, once Cas13 locates and cuts its specified target, the enzyme goes into overdrive, indiscriminately cutting other RNA nearby. To create SHERLOCK, the team harnessed this off-target activity and turned it to their advantage, engineering the system to be compatible with both DNA and RNA.

SHERLOCKs diagnostic potential relies on additional strands of synthetic RNA that are used to create a signal after being cleaved. Cas13 will chop up this RNA after it hits its original target, releasing the signaling molecule, which results in a readout that indicates the presence or absence of the target.

Multiple targets and increased sensitivity

The SHERLOCK platform can now be adapted to test for multiple targets. SHERLOCK initially could only detect one nucleic acid sequence at a time, but now one analysis can give fluorescent signals for up to four different targets at once meaning less sample is required to run through diagnostic panels. For example, the new version of SHERLOCK can determine in a single reaction whether a sample contains Zika or dengue virus particles, which both cause similar symptoms in patients. The platform uses Cas13 and Cas12a (previously known as Cpf1) enzymes from different species of bacteria to generate the additional signals.

SHERLOCKs second iteration also uses an additional CRISPR-associated enzyme to amplify its detection signal, making the tool more sensitive than its predecessor. With the original SHERLOCK, we were detecting a single molecule in a microliter, but now we can achieve 100-fold greater sensitivity, explained co-first author Omar Abudayyeh, an MIT graduate student in Zhangs lab at Broad. Thats especially important for applications like detecting cell-free tumor DNA in blood samples, where the concentration of your target might be extremely low. This next generation of features help make SHERLOCK a more precise system.

The authors have made their reagents available to the academic community through Addgene and their software tools can be accessed via the Zhang lab website and GitHub.

This study was supported in part by the National Institutes of Health and the Defense Threat Reduction Agency.

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Researchers advance CRISPR-based tool for diagnosing disease …

Researchers use CRISPR to detect HPV and Zika

The first study comes from the lab of CRISPR pioneer Jennifer Doudna. Her team discovered that a CRISPR system different from the CRISPR-Cas9 one we’re used to hearing about can not only snip away specific bits of double-stranded DNA, but can then also cut single-stranded DNA that’s near it. After they uncovered this ability of CRISPR-Cas12a, they used it to detect two common types of HPV. Once their CRISPR-Cas12a system detected HPV DNA in infected cells, it cleaved a another piece of DNA that then released a fluorescent signal, providing a visual sign of the presence of HPV. The researchers dubbed the system DETECTR and The Verge reports that it takes around an hour to work and costs less than a dollar.

The lab of another CRISPR pioneer, Feng Zhang, has now improved on a previous system it developed last year. SHERLOCK, as it’s called, can detect specific bits of DNA and RNA to determine whether viruses like Zika or dengue are present in a blood sample, identify mutations in tumor DNA and spot the presence of harmful bacteria. In their latest study, the research team describes SHERLOCK version 2.0, which is not only over three times as sensitive as the first version, but can also detect both Zika and dengue in the same sample. Their system uses several CRISPR enzymes, including Cas13 and Csm6, and can be loaded onto a paper strip, making it incredibly easy to use. You can see examples of the strips in the GIF below. Jonathan Gootenberg, one of the authors of the study, told The Verge, “The fact that we can put all these different enzymes into a single tube and have them not only play nice with each other, but also tell us information we couldn’t get otherwise — that is really spectacular and it speaks to a lot of the power of biochemistry.”

Lastly, Harvard University’s David Liu published a study showing that CRISPR can be used to track certain ongoings in a cell. Seeing what a cell has been exposed to in the past has been a rather hard thing to do, but CRISPR systems provide a way for researchers to do just that. Liu’s team used CRISPR in two different ways to record when a cell was exposed to certain chemicals. In the first, CRISPR was used to snip bits of DNA called plasmids if it came in contact with a particular chemical, such as an antibiotic or a nutrient. By comparing the ratio of the plasmid types that were destroyed by CRISPR to other, similar plasmids that were left alone, the researchers were able to determine just how often the cells were exposed to those chemicals. Another version of the system changed individual letters, or bases, of DNA rather than snipping plasmids and the team was able to determine when cells were exposed to antibiotics, nutrients, viruses and light by examining those changes in the DNA bases.

While all three of these systems need further development before they can be used outside of the lab, they show that CRISPR has quite a lot of uses, beyond just treating disease. The technology is incredibly versatile and we’re sure to see even more applications going forward.

Image: Zhang Lab, Broad Institute of MIT and Harvard

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Researchers use CRISPR to detect HPV and Zika

CRISPR Gene Editing And 3 Biotech Companies Blaze New Path To …

Imagine editing one gene and curing a debilitating disease.Three small biotech companies with combined annual sales of less than $50 million Crispr Therapeutics (CRSP), Intellia Therapeutics (NTLA) and Editas Medicine (EDIT) say that soon could be a reality.

X All three biotech stocks went public in 2016 to bet big on a simple premise: Altering specific genes can create curative medicines.An estimated 5,000 diseases could be cured by changing one targeted gene, says former Intellia Chief Executive Nessan Bermingham.

The World Health Organization has a higher estimate for what are known as monogenic diseases and says it’s actually north of 10,000.

“People have been talking about (personalized medicine) for 20 years and yet we’ve never had a system to allow us to do it before,” Bermingham told Investor’s Business Daily before stepping down from his role on Dec. 31.”And for the first time ever, we actually have a system to do it and that system would be based on your personalized genome.”

That system is known as CRISPR, and it’s where Crispr, Intellia and Editas are putting their chips. It’s a cheaper and faster gene editing method and, according to Bermingham, the key to advancing personalized medicine. Some analysts think CRISPR technology could provide the platform for the next generation of giant biotech companies.

CRISPR the technology not to be confused with Crispr Therapeutics, the company builds on a project that sequenced the human genome. The first map cost $2.7 billion and was completed in 2003.

Since then, the cost to map an individual’s genome has dropped precipitously and could come down to just hundreds of dollars in the next few years, Bermingham says. Large-data analytics also have a part to play in sifting through the genome.

IBD’S TAKE:Biotech companies account for a large share of recent IPO stocks, yet investing in them before they have profits or sales can be risky. Learn to identify the best IPOs and how to trade themfor potential big gains.

When the first human genome was mapped, investigators were “absolutely horrified” to find just 20,000 genes in the human body that code proteins, Bermingham says. That was down from estimates of 100,000. Essentially, these protein-coding genes serve as words in the genetic language.

Investigators also found regions of DNA that were initially thought to have no purpose. These were controversially called “junk DNA” that does not code protein. But, as it turns out, these sequences do have a key purpose in regulating the expression of genes.

All together, the better understanding of the human genome has allowed these biotech companies to utilize CRISPR, an acronym for the technology known asClustered Regularly Interspaced Short Palindromic Repeats.

There is a caveat, however. In January, a paper published by bioRxiv said there may be evidence that human immune systems may fight off the major form of genome editing that uses an enzyme called Cas9, thus rendering the science ineffective. The paper, however, has yet to be peer reviewed.

The process, developed at various universities, essentially uses specialized strands of DNA thatact as molecular “scissors.” Those scissors are capable of editing other DNA at specific points, and allow biotech companies to edit, add or remove faulty genes responsible for diseases.

There are varying types of scissors. Crispr, Intellia and Editas are using the Cas9 CRISPR technology, ARK Invest analyst Manisha Samy told IBD. She estimates Cas9 can reach 70%-80% of the human genome. Developing new scissors can expand the reach into more genes and diseases, she says.

Gene editing isn’t new, she adds. Older techniques called TALENs and zinc finger nucleases have been around for some time. Notably, biotech companyBluebird Bio (BLUE) is using a variation of TALENs, and Sangamo Therapeutics (SGMO) is using a method of zinc finger nucleases.

She likens CRISPR technology to a word processor.

“We think CRISPR gene editing is analogous to a DNA word processor with two functions: find and delete,” she said in a January 2017 report. “In addition, scientists are working on a rudimentary paste function, allowing CRISPR to insert appropriate DNA code to repair mutations.”

Older technologies used by biotech companies are more like old-fashioned typewriters, requiring actual cutting and pasting, she says. CRISPR technology is also cheaper and easier to use than TALENs and zinc fingers, says JMP Securities analyst Mike King.

“What’s so powerful about CRISPR is it’s so easy to use,” he told IBD. “High school students are doing experiments in the biology lab to knock out genes. Zinc fingers takes a lot of talent and time. You have to fiddle with them a lot. The systems created under CRISPR are quite robust.”

In January, bioRxiv an online archive and distribution service for unpublished reports in the life sciences field published a paper casting doubt on the durability of CRISPR gene therapy over time, suggesting the body could build an immunity to it. Analysts and biotech companies are not worried, however, saying either this is a nonissue or there’s time for the science to catch up.

Many companies working in CRISPR are doing so using the Cas9 enzyme, short for CRISPR associated protein 9. Cas9 is derived from two bacteria that cause infections in humans at high rates, meaning some immune systems could have developed immunities to them.

Would CRISPR gene editing, using that enzyme, work in those patients?

It depends, Crispr Therapeutics said in a follow-up email to IBD. It’s important to note the lead investigator and writer on the bioRxiv paper wasMatthew Porteus, a scientific founder and advisory board member for Crispr Therapeutics.

When the gene editing is done ex vivo, or outside the body, the Cas9 enzyme is degraded and, therefore, essentially gone by the time the cells are reintroduced to the patient, Crispr told IBD.

For in vivo applications, when gene editing is done inside the body, Crispr Therapeutics says it uses several approaches to ensure transient expression of the Cas9 enzyme. Because of that, “we do not expect pre-existing immunity to Cas9 to cause any issues,” the firm said.

Ark’s Samy also noted that other enzymes are in use. Editas is also using the Cpf1 enzyme. This enzyme is derived from other bacteria and could overcome some of the immunity challenges involving Cas9.

Intellia told IBD in a follow-up email that in clinical testing, its delivery system for treatment in rodents and non-human primates has yet to falter. Further, Intellia notes it’s using an advanced form of Cas9 and none of the donors had a pre-existing immunity in its study.

The data are still early. Editas has done its own work in immune responses to CRISPR genome editing and will present a paper in the future, JMP’s King said in a Jan. 8 note to clients. Management has indicated it found immune responses to be “much lower” than those reported in the other paper.

“Immune responses are not uncommon,” Samy said. “Scientists have worked for decades on evading immune recognition. There are numerous workarounds that can be implemented to reduce any potential side effects with Cas9 and we have proved this in a number of other therapeutic modalities.”

Among the biotech companies, Crispr Therapeutics is ahead of the competition from a regulatory standpoint. On Dec. 7, the firm submitted its first application for a clinical trial testing its gene therapy, known as CTX001, in a blood disorder known as beta thalassemia.

The company is working with Vertex Pharmaceuticals (VRTX) in beta thalassemia, as well as sickle cell disease. The therapies are part of Crispr’s ex vivo programs, where gene editing is done on cells outside the body before they are reintroduced to the patient. Crispr is also looking at in vivo therapies for the liver, muscles and lungs.

According to a Crispr news release, the trial is set to begin in Europe in 2018 in adult patients. This is expected to be the first in-human trial of a gene editing treatment based on CRISPR technology. Crispr also plans to file an application to begin testing for CTX001 in treating sickle cell disease in the U.S. in 2018.

Intellia also has in vivo and ex vivo programs in gene editing, and also is working in sickle cell disease. It’s furthest along in a partnership with Regeneron Pharmaceuticals (REGN) for a therapy to treat what’s known as transthyretin amyloidosis, a condition characterized by the buildup of abnormal protein deposits throughout the body.

Alnylam Pharmaceuticals (ALNY) and Ionis Pharmaceuticals (IONS) also are working separately to treat the disease using different methods called RNA interference and antisense technology, respectively.

Meanwhile, Editas is working on an injected treatment for an inherited eye disease known as Leber congenital amaurosis, which is characterized by severe loss of vision at birth. It is also using gene editing in sickle cell disease and beta thalassemia.

Intellia and Editas also are expected to start in-human trials in 2018, analysts say, though Intellia has not said when it will begin testing.

Regulators are getting more comfortable with the idea of gene editing, Crispr Therapeutics President Sam Kulkarni told IBD. The benefit of gene editing and potential trouble with it is that it’s meant to be a permanent fix. The biotech companies are working to ensure they hit a bull’s-eye every time out.

“We’ve shown you we can make this edit and it’s done in a precise fashion using (targets the industry calls) molecular ZIP codes,” he said. “We eliminate edits happening outside places you want them to happen. And we manufacture these in a high-quality fashion, understanding the pharmacology.”

Both Kulkarni and Intellia’s Bermingham who was succeeded byJohn Leonard, a former AbbVie (ABBV) executive say there’s room for all three big players in the group.

Sizing the market is a challenge, ARK’s Samy says. No matter how you slice it, the numbers are big and a lot will depend on which diseases companies target and how they set pricing.

If CRISPR is able to address all monogenic diseases diagnosed each year, that’s a $75 billion market globally, she says. Addressing all these diseases for people already living with diagnoses would be a $2 trillion market.

“One product is not going to cure everything,” she said. “Whenever you’re seeing volatility between these three main CRISPR companies, it doesn’t really make sense because there’s room for all of them and more when it comes to CRISPR.”

Kulkarni says it’s unlikely the market will remain at just three publicly traded biotech companies with CRISPR technology in the long run. The technology is just that remarkable.

“Once in a lifetime may be a little bit of a stretch, maybe not,” he said. “But it’s definitely a once in a generation type of advance in the field. The last time this kind of excitement happened in the biotech field was when antibodies were applied as therapeutic modalities. On the basis of that, technology companies like Genentech (now owned byRoche (RHHBY)) were created.”

He added: “Here we have the basis of a CRISPR platform to create the next big biotech giants.”

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CRISPR Gene Editing And 3 Biotech Companies Blaze New Path To …

Chinese scientists already used Crispr gene editing on 86 …

China is taking the lead in the global race to perfect gene therapies.

Scientists have genetically engineered the cells of at least 86 cancer and HIV patients in the country using Crispr-Cas9 technology since 2015, the Wall Street Journal reports (paywall). Although no formal scientific papers have been written about these experiments, doctors told journalists at the WSJ that some patients have improved. There have also been least 15 deaths, seven of which were in one trial. Scientists report all of these deaths were related to patients previous conditions and not Crispr treatment.

These therapies, which involved taking the immune cells from hospital patients, editing the cells, and transfusing them back into the body, are the first to use Crispr-Cas9 in living humans.

In 2013 scientists first used (paywall) Crispr on on human DNA, and in 2017, US scientists at Oregon Health & Science University reported using the technology to edit human embryos. (The embryos were not allowed to develop further.) It took two years for the Oregon team to receive ethical approval for their experiment. It took the same amount of time for the University of Pennsylvania hospital and the US Food and Drug Administration to give Penn researchers the go-ahead to test a Crispr-based therapy on 18 cancer patients. That trial is expected to begin later this year. Scientists at the Cambridge, Massachusetts-based Crispr Therapeutics also hope to start phase I clinical trials using Crispr to treat patients with a genetic disorder called beta-thalassemias.

Crispr trials on humans have been relatively slow to develop in the US and UK in part due to concerns over how the risk of the procedure is communicated to patients. The Penn scientists first had to consult with an advisory board from the National Institutes of Health set up specifically to evaluate the potential risks and benefits of Crispr therapies, then get approval from the US Food and Drug Administration.

The FDA approved three gene therapies for treatment in 2017, none of which use Crispr. Two of these therapies treat late-stage forms of cancer, and both rely on editing the patients immune cells. The third, which targets a rare form of childhood blindness, works by modifying cells in the eye.

The Chinese ministry of health has to approve all gene-therapy clinical trials in China, but these regulations appear relatively relaxed. According to the WSJ, at Hangzhou Cancer Hospital, for example, a proposal to test a cancer treatment that modifies patients immune cells was approved in a single afternoon. One member of the hospitals approval committee told the WSJ that she did not really understand the science laid out for her in a 100-page document, but was told that the side effects were mild. This was enough for her to give it the go-ahead.

The truth, though, is that there is a dearth of data on the safety of Crispr on humans, and many scientists in the field are concerned that the treatment may cause unintended mutations or may not work at all.

If any of these Crispr treatments are proven successful under scientific scrutiny, theyd be the first of their kind.

Correction: An earlier version of this article stated that about half of the deaths in Crispr trials were related to the gene therapy. It has been corrected to reflect that doctors say all of the deaths in the Crispr trials were related to patients previous conditions.

Read this next: A highly successful attempt at genetic editing of human embryos has opened the door to eradicating inherited diseases

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Crisprs Next Big Challenge: Getting Where It Needs to Go | WIRED

Your DNA is your bodys most closely guarded asset. To reach it, any would-be-invaders have to get under your skin, travel through your bloodstream undetected by immune system sentries, somehow cross a cell membrane, and finally find their way into the nucleus. Most of the time, thats a really good thing. These biological barriers prevent nasty viruses from turning your cells into disease-making factories.

But theyre also standing between patients with debilitating genetic diseases and their cures. Crispr, the promising new gene editing technology, promises to eradicate the world of human sufferingbut for all the hype and hope, it hasnt actually cured humans of anything, yet. Medical researchers have the cargo, now they just have to figure out the delivery route.

The first US trials of Crispr safety are set to begin any day now, with Europe expected to follow later this year. Chinese scientists, meanwhile, have been testing Crispr humans since 2015, as The Wall Street Journal recently reported, with mixed success. These first clinical forays involve removing cells from patients bodies, zapping them with electricity to let Crispr sneak in, then infusing them back into their bodies, to either better fight off cancer or to produce a missing blood protein. But that wont work for most rare genetic diseasesthings like cystic fibrosis, Duchennes muscular dystrophy, and Huntingtons. In the 34 trillion-cell sea that is your body, an IV bag full of Crisprd cells simply wont make a dent.

This is the same problem that has plagued the stop-and-go field of gene therapy for nearly three decades. Traditional gene therapy involves ferrying a good copy of a gene inside a harmless virus, and brute-forcing it into a cells DNA. Crisprs cutting action is much more elegant, but its bulk and vulnerability to immune attacks make it just as difficult to deliver.

The challenge is getting gene editors to the right place at the right time in the right amount, says Dan Anderson, an MIT chemical engineer and one of the scientific founders of Crispr Therapeutics. Thats a problem people have been working on for a long time. As of today there certainly is no one way to cure every disease with a single delivery formulation.

And its unlikely there will be anytime soon. So for now, most Crispr companies are taking more of a whatever works approach, borrowing mostly from gene therapys few success stories. One of those is a small, harmless helper virus called AAV, well-suited for carrying genetic instructions into a living cell. AAV wont make you sick, but it can still sneak into your cells and hijack their machinery, making them a perfect Trojan horse in which to put good stufflike a correct copy of a gene, or instructions for how to make the protein-RNA pair that forms the Crispr complex. Crisprs instructions are quite long, so they often cant fit inside one virus.

But once you get around that, theres an even bigger downside to AAV; once it ferries Crispr inside a cell, theres no good way to control its expression. And the longer Crispr hangs around, the greater the chance it could make unwanted cuts.

Delivering Crispr into the cell directly, as opposed to teaching the cell to build it, would provide more control. But doing that means enveloping the unwieldy, charged protein complex in a coating of fat particlesone that can simultaneously shield it from the immune system, get it across a cell membrane, and then release it to do its cutting work unencumbered. Although the technology is improving, its still not very efficient.

The big threeCrispr Therapeutics, Editas Medicine, and Intellia Therapeuticsas well as the latest newcomer, Casebia, are all investing in AAV and lipid nanoparticles, and testing both for their first rounds of treatment. Were leveraging existing delivery technologies, while exploring and developing the next generation, says Editas CEO Katrine Bosley. We will use whatever works best for a given target.

But industry isnt the only one feeling the urgency. This week the National Institutes of Health announced it will be awarding $190 million in research grants over the next six years, in part to push gene editing technologies into the mainstream. The focus of the Somatic Cell Genome Editing program is to dramatically accelerate the translation of these technologies to the clinic for treatment of as many genetic diseases as possible, NIH Director Francis Collins said in a statement Tuesday. Which could encourage some of the more exotic, experimental delivery systems out in the research worldstrategies like Crispr-covered gold beads, yarn-like ball structures called DNA nanoclews, and shape-shifting polymers to get the editor where it needs to go.

In October, UC Berkeley researchers Kunwoo Lee, Hyo Min Park, and Nirhen Murthy used those gold nanoparticles to repair the muscular dystrophy gene in mice. Theyre now expanding that work in a startup the trio cofounded called GenEdit. They plan to develop a suite of nanoparticle delivery vehicles optimized to different tissues, starting with muscles and the brain. Then theyll partner with the folks making the Crispr payloads. That will make it the first company devoted solely to Crispr delivery. The gene editing world is filling up with products to deliverbut even Amazon needs UPS.

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Crisprs Next Big Challenge: Getting Where It Needs to Go | WIRED

New CRISPR method could take gene editing to the next level

Remove and replace

Science / Alamy Stock Photo

By Michael Le Page

The CRISPR genome-editing method may just have become even more powerful. Uri David Akavias team at McGill University in Canada has managed to repair mutations in 90 per cent of target cells using CRISPR the best success rate yet.

The CRISPR approach is very good at disabling genes, but using the technique to fix them is much harder, because it involves replacing a faulty sequence with another. This typically works in less than 10 per cent of target cells.

To make the process more efficient, Akavias team physically linked the replacement DNA with the CRISPR protein that finds and cuts the faulty sequence. This ensures that the replacement DNA is there ready to be slotted in once the cut is made. Weve taped the [replacement] text to the scissors, says Akavia.

The team also used a polymer

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Obstacle to Using CRISPR in Humans? The Immune System – The …

2018 is supposed to be the year of CRISPR in humans. The first U.S. and European clinical trials that test the gene-editing tool’s ability to treat diseasessuch as sickle-cell anemia, beta thalassemia, and a type of inherited blindnessare slated to begin this year.

But the year has begun on a cautionary note. On Friday, Stanford researchers posted a preprint (which has not been peer reviewed) to the website biorXiv highlighting a potential obstacle to using CRISPR in humans: Many of us may already be immune to it. Thats because CRISPR actually comes from bacteria that often live on or infect humans, and we have built up immunity to the proteins from these bacteria over our lives.

Its the first time this concern has been aired so publicly, and the preprint kicked off something of a firestorm. We had no anticipation it would be picked up so broadly on social media. I dont even have a Twitter account. I just heard this from others, says Matthew Porteus, a pediatrician and stem-cell researcher at Stanford who led the study and is working on a clinical trial for sickle-cell anemia.

Not all CRISPR therapies in humans will be doomed. We dont think this is the end of the story. This is the start of the story, says Porteus. There are likely ways around the problem of immunity to CRISPR proteins, and many of the early clinical trials appear to be designed around this problem.

Porteus and his colleagues focused on two versions of Cas9, the bacterial protein mostly commonly used in CRISPR gene editing. One comes from Staphylococcus aureus, which often harmlessly lives on skin but can sometimes causes staph infections, and another from Streptococcus pyogenes, which causes strep throat but can also become flesh-eating bacteria when it spreads to other parts of the body. So yeah, you want your immune system to be on guard against these bacteria.

The human immune system has a couple different ways of recognizing foreign proteins, and the team tested for both. First, they looked to see if people have molecules in their blood called antibodies that can specifically bind to Cas9. Among 34 people they tested, 79 percent had antibodies against the staph Cas9 and 65 percent against the strep Cas9.

Then, they looked to see if a particular type of immune cells called T cells can recognize the Cas9 proteins. This time they studied T cells from 13 healthy adults. Six of themor 46 percentreacted to the staph Cas9. None of them did against the strep Cas9.

The Stanford team only tested for preexisting immunity against Cas9, but anytime you inject a large bacterial protein into the human body, it can provoke an immune response. After all, thats how the immune system learns to fight off bacteria its never seen before. (Preexisting immunity can make the response faster and more robust, though.)

In statements to The Atlantic, three of the leading companies in CRISPR human therapyEditas Medicine, CRISPR Therapeutics, and Intellia Therapeuticsall downplayed the new findings, citing various ways their therapies could around the immune system. (Porteus is a scientific founder of CRISPR Therapeutics, though this study was performed independently.) A September 2017 presentation from a scientist at Editas Medicine also detailed some of the ways to test for immune reactions to CRISPR, anticipating a potential problem.

Here are some possible strategies to get around the immune system that are being discussed and tested:

Only use CRISPR outside of the body: Instead of delivering CRISPR/Cas9 into the body, you take cells out of the body, use CRISPR to edit their genes in a lab, and return Cas9-free cells. This is the strategy pursued by CRISPR Therapeutics for the inherited blood disorder thalassemia and in various trials using CRISPR to modify immune cells to attack cancer.

Only use CRISPR in places the immune system cannot reach: Some sites of the body are immunoprivileged, meaning the immune system cant really attack invaders there. The eye, which Editas Medicine is targeting for inherited blindness, is one of those sites.

Modify Cas9 or use a different CRISPR protein altogether: It may be possible to redesign Cas9 to hide it from the immune system or to find other bacterial proteins that can do the job of Cas9 without provoking the immune response. Many different bacteria have CRISPR systems. We already have lots of Cas enzymes and could get many more, George Church, a geneticist at Harvard and a founding scientific advisor of Editas, wrote in an email.

Express Cas9 only transiently:Once Cas9 has made its edit, it doesnt need to stick around. A spokesperson for Intellia noted that its still unclear how the immune system responds to continuous versus transient expression of Cas9. The company says its lipid-nanoparticle delivery system can get cells to make Cas9 only transiently, but enough for the editing to happen in rodents and non-human primates.

The one type of therapy where immune response may be most dangerous and unavoidable is when Cas9 is produced for a prolonged period of time in a non-immunoprivileged site. In the liver, for instance, the immune system could end up attacking the Cas9-making liver cells.

The danger of the immune system turning on a patients body hangs over a lot of research into correcting genes. In the late 1990s and 2000s, research into gene therapy was derailed by the death of 18-year-old Jesse Gelsinger, who died from an immune reaction to the virus used to deliver the corrected gene. This is the worst-case scenario that the CRISPR world hopes to avoid.

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CRISPR hits a snag: Our immune systems may attack the treatment

A

new paper points to a previously unknown hurdle for scientists racing to develop therapies using the revolutionary genome-editing tool CRISPR-Cas9: the human immune system.

In a study posted Friday on the preprint site bioRxiv, researchers reported that many people have existing immune proteins and cells primed to target the Cas9 proteins included in CRISPR complexes. That means those patients might be immune to CRISPR-based therapies or vulnerable to dangerous side effects the latter being especially concerning as CRISPR treatments move closer to clinical trials.

But researchers not involved with the study said its findings, if substantiated, could be worked around. (Papers are posted to bioRxiv before being peer-reviewed.) Many of the first planned CRISPR clinical trials, for example, involve removing cells from patients, fixing their DNA, and then returning them to patients. In that case, its possible that there will be few or no CRISPR proteins remaining for the immune system to detect.

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They also noted that scientists are already studying other types of CRISPR that use different proteins, which could stave off the immune responses.

At the end of the day, Im not that concerned about it, said Daniel Anderson of the Massachusetts Institute of Technology, who has studied the delivery of CRISPR therapies and who was not involved with the new study. But we want to do some experiments to learn more.

The new study should not put the brakes on developing CRISPR therapies, agreed Dr. Matthew Porteus of Stanford, a senior author of the paper and who is himself at work on a CRISPR-based therapy for sickle cell disease. But he said he and his colleagues investigated the immune issues because he felt they were being overlooked as the excitement around CRISPR grew.

Like any new technology, you want to identify potential problems and engineer solutions for them, Porteus said. And I think thats where were at. This is an issue that should be addressed.

(Porteus and Anderson are both scientific founders of CRISPR Therapeutics, one of the most prominent companies exploring CRISPR-based therapies.)

CRISPR has gained fame in recent years as researchers have deployed it to correct an array of disease-causing mutations in cells in the lab and in animal models, with hopes that the same results can be achieved in people. There are different types of CRISPR systems, but the most well known is dubbed CRISPR-Cas9; it includes Cas9 proteins that cut DNA so that it can be edited. Cas9 proteins come from bacteria.

For the study, the researchers decided to check for immune signals against two of the most common types of Cas9 proteins used, those from the bacteria S. aureus (called SaCas9) and those from S. pyogenes (called SpCas9). In their samples of blood from 22 newborns and 12 adults, the scientists found that 79 percent of donors had immune proteins, called antibodies, against SaCas9, and 65 percent had antibodies against SpCas9.

The researchers then searched for immune cells called T cells. They discovered that about half of the donors had T cells that specifically targeted SaCas9, so that if the immune cells detected that protein on the surface of a cell, they would rally a response to try to destroy it. The researchers did not find anti-SpCas9 T cells, though they said the cells might still have been present.

Its not surprising so many of the donors had antibodies and T cells against the Cas9 proteins, experts said. That simply means that those people had been exposed to the bacteria containing the proteins in the past, and other studies have found that, at any given time, 40 percent of people are colonized by S. aureus and 20 percent of schoolchildren have S. pyogenes. The bacteria only sometimes cause disease.

But what then does that previous exposure mean for our receptiveness to CRISPR therapies?

A lot remains unclear, Porteus said. Its not known how severe the immune response would be, and whether it would trigger a dangerous inflammatory attack or just render the treatment useless.

Experts also said that perhaps the immune responses could be avoided. If the CRISPR complex does its editing after the cells are removed from the patient whats called ex vivo or in a place like the eye that is isolated from the immune system, then the antibodies and T cells might not detect any Cas9 proteins. Even in in vivo therapies in which CRISPR complexes would be ferried into cells in a patients body much depends on what kind of delivery system is used and whether the Cas9 proteins become expressed on the outside of the cells in which the editing is taking place.

Porteus said he and his team decided to post the paper on bioRxiv because they wanted CRISPR researchers to start thinking now about possible immune system challenges. The team has also submitted the paper to a journal for peer review and publication.

As a cautionary tale about the importance of asking these questions now, Porteus pointed to what happened with gene therapy in 1999. In that case, a patient in a trial died after an immune system attack, likely because he had preexisting antibodies against a virus used as part of the therapy. The death led to years lost in gene therapy development, experts say. (Patients who have preexisting antibodies to viruses used in gene therapies are now generally excluded from trials.)

I would hate to see the field have a major setback because we didnt address this potential issue, Porteus said. We should learn from that.

Roland Herzog, a gene therapy expert at the University of Florida, agreed that the hype around CRISPR meant that possible immune issues were not being given enough credence.

I suspect that the field has not been aware of it sufficiently, he said. Its not a show stopper, he added about the paper, but the field needs to know about this, that its a potential problem that they need to work around or fix.

One possible fix is simply using a different protein or enzyme in the CRISPR complex, one that doesnt come from such common bacteria. If people havent been exposed to the bacterial protein previously, then they wont have specific antibodies or T cells ready to attack.

New Cas editing enzymes are being described all the time from bacterial species that are not human pathogens (and so there would be no chance to develop the pre-existing antibodies), Jacob Corn, of the University of California, Berkeley, who was not involved with the new paper, wrote in an email. I also know some people have already been working on making Cas enzymes that would be invisible to the immune system.

He added: The field moves very fast!

General Assignment Reporter

Andrew is a general assignment reporter at STAT.

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CRISPR hits a snag: Our immune systems may attack the treatment

In 2018, We Will CRISPR Human Beings – gizmodo.com

Ever since 2012, when researchers first discovered that bacterial immune systems could be hijacked to edit DNA in living creatures, CRISPR has been hailed as a maker of revolutions. This was the year that prediction felt like it was starting to come true. U.S. scientists used the CRISPR gene editing technique to treat a common genetic heart disease in a human embryo. Many morediseases were successfully treated in mice using CRISPR. Hell, a particularly enthusiastic biohacker even spontaneously injected himself with muscle-growth genes while giving a talk at a conference.

But if 2017 was the year that the potential of CRISPR began to come into focus, 2018 may be the year that potential begins to be realized.

Next year, the first human trials of CRISPR-based treatments in the U.S. and Europe are slated to begin.

This month, biotech firm CRISPR Therapeutics became the first to submit a clinical trial application to European regulators. Tests are set to begin next year for its therapy that combines CRISPR gene editing and stem cell therapy to treat the blood disorder beta thalassemia. CEO Samarth Kulkarni told Gizmodo that the company also plans to file an application to conduct a clinical trial using a similar therapy to treat sickle cell disease in the first half of 2018. In 2018, the first human is going to get dosed with CRISPR in the clinic, Kulkarni told Gizmodo. And were going to be the first ones to do it.

Both disorders are genetic, caused by mutations to the genes that produce hemoglobin, a protein essential to ensuring that red blood cells ferry oxygen throughout the body. Without that oxygen, people can suffer from severe anemia, developmental delays, damage to organs, and pulmonary hypertension. The idea is extract stem cells from patients bone marrow and correct the faulty genes with CRISPR, a gene-editing technique that allows scientists to cut and paste tiny snippets of genetic code. Then those edited cells would be infused back into the body, where they would multiply, eventually outnumbering the diseased cells. Sickle cell disease and beta thalassemia are good candidates for CRISPR because in many cases, they are caused by a mutation to one single DNA letter.

At Stanford, a different spin on using CRISPR to treat sickle cell disease is also moving toward clinical trials. Matthew Porteus, who heads the research, said that his group expects to file a clinical trial application with the FDA by the end of 2018 and begin trials in 2019. Our New Years resolution for 2018 is to gather the data so we can file a [trial application] by the end of the year, so we can start a clinical trial in 2019, Porteus told Gizmodo. We just need to check off all the boxes.

Chinese scientists, meanwhile, used CRISPR for the first time on a human in 2016, and conducted a second human trial this year, setting off a biomedical duel between the U.S. and China and sparking concerns that the trials were irresponsibly premature. The first U.S. human CRISPR trial was slated to begin this summerat the University of Pennsylvania, after receiving a regulatory stamp of approval to proceed last year. It is unclear what has caused that trials delay.

Porteus said that he expects 2018 will bring many more preclinical studies demonstrating how CRISPR might be used to treat different diseases. In 2017, there were several such studies, addressing devastating diseases and conditions such as Huntingtons disease, Lou Gherigs disease, and an inherited form of hearing loss in mice.

There is going to be a lot of behind-the-scenes work of turning those into a real clinical protocol, Porteus said. He also predicted 2018 will see applications for more clinical trials, though most likely ones the involve simply deleting a problematic gene rather than correcting it.

George Church, the famed Harvard geneticist, told Gizmodo that he expects CRISPR will get much more precise in the coming year. He also expects an uptick in research on how to use CRISPR to solve problems that dont have other good solutions, like eliminating zoonotic diseases such as Lyme disease and malaria by using whats known as a gene drive to alter the DNA of wild species, or even growing transplantable organs in pigs.

The MIT synthetic biologist Kevin Esvelt said he expects there to be more gene therapy progress using a brand-new CRISPR technique that relies on base editing, or chemically altering a single letter of DNA rather than using CRISPR to actually cut through DNAs double helix to change it. The only prediction Im absolutely confident of making is that 2018 will see CRISPR continue to markedly accelerate research, both by simplifying previously difficult tasks and by making it possible to conduct experiments we could never previously contemplate, Esvelt said. Beyond that, theres no telling.

Hank Greely, a bioethicist at Stanford, told Gizmodo that he expects to see advancement with CRISPR outside of biomedicine. Among his predictions: Several groups will come up with completely new and unexpected uses for CRISPR, he said. And someone, somewhere will do a gene-drive trial in a controlled but non-laboratory environment.

Greely also predicts that CRISPR inventors will win a Nobel prize. (Though theres no telling which of CRISPRs inventorsits a bitterly disputed claim that award would go to.)

There are still significant hurdles, though, to reaching a future in which molecular cutting and pasting can act as a one-time cure-all for any genetic disease. For one, treating diseases that require editing DNA while its still inside the human body, such as amyotrophic lateral sclerosis,is a lot harder (and riskier) than removing cells, editing them in a lab, and putting them back into the body, as researchers will do in the trials slated to start in the next two years. But addressing many diseases will require whats known as in-vivo treatment. And scientists are still working to figure out the best way to deliver therapies inside the body effectively.

For ex-vivo treatments, the limit now is just whether we can do the work. I dont think there are obvious technical challenges. We just need to move into the clinic and test them out, Porteus said. For for in-vivo treatments, there is a lot of room for improvement.

Greely said that while some CRISPR clinical trials will start soon, they wont wrap up in 2018. Even when science is moving at a breakneck speed, like it is with CRISPR, it still tends to move more slowly than we wish it would.

And we may also realize that the high-tech solution is not always the best option.

Harvard geneticist Church said CRISPR may be due for a reality check. For example, he said, families may decide to undergo genetic counseling before having kids to assess their risk of passing on genetic diseases, rather than having children and treating them with CRISPR therapies likely to be $1 million per dose.

Whatever is in store for 2018, its important to remember that in the realm of science, progress is never a straight line.

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In 2018, We Will CRISPR Human Beings – gizmodo.com

Crispr Isnt Enough Any More. Get Ready for Gene Editing 2.0

In fewer than five years, the gene-editing technology known as Crispr has revolutionized the face and pace of modern biology. Since its ability to find, remove, and replace genetic material was first reported in 2012, scientists have published more than 5,000 papers mentioning Crispr. Biomedical researchers are embracing it to create better models of disease. And countless companies have spun up to commercialize new drugs, therapies, foods, chemicals, and materials based on the technology.

Usually, when weve referred to Crispr, weve really meant Crispr/Cas9a riboprotein complex composed of a short strand of RNA and an efficient DNA-cutting enzyme. It did for biology and medicine what the Model T did for manufacturing and transportation; democratizing access to a revolutionary technology and disrupting the status quo in the process. Crispr has already been used to treat cancer in humans, and it could be in clinical trials to cure genetic diseases like sickle cell anemia and beta thalassemia as soon as next year.

But like the Model T, Crispr Classic is somewhat clunky, unreliable, and a bit dangerous. It cant bind to just any place in the genome. It sometimes cuts in the wrong places. And it has no off-switch. If the Model T was prone to overheating, Crispr Classic is prone to overeating.

Even with these limitations, Crispr Classic will continue to be a workhorse for science in 2018 and beyond. But this year, newer, flashier gene editing tools began rolling off the production line, promising to outshine their first-generation cousin. So if you were just getting your head around Crispr, buckle up. Because gene-editing 2.0 is here.

Crisprs targeted cutting action is its defining feature. But when Cas9 slices through the two strands of an organisms DNA, the gene-editor introduces an element of risk. Cells can make mistakes when they repair such a drastic genetic injury. Which is why scientists have been designing ways to achieve the same effects in safer ways.

One approach is to mutate the Cas9 enzyme so it can still bind to DNA, but its scissors dont work. Then other proteinslike ones that activate gene expressioncan be combined with the crippled Cas9, letting them toggle genes on and off (sometimes with light or chemical signals) without altering the DNA sequence. This kind of epigenetic editing could be used to tackle conditions that arise from a constellation of genetic factors, as opposed to the straightforward single mutation-based disorders most well-suited to Crispr Classic. (Earlier this month, researchers at the Salk Institute used one such system to treat several diseases in mice, including diabetes, acute kidney disease, and muscular dystrophy.)

Other scientists at Harvard and the Broad Institute have been working on an even more daring tweak to the Crispr system: editing individual base pairs, one at a time. To do so, they had to design a brand-new enzymeone not found in naturethat could chemically convert an A-T nucleotide pairing to a G-C one. Its a small change with potentially huge implications. David Liu, the Harvard chemist whose lab did the work, estimates that about half of the 32,000 known pathogenic point mutations in humans could be fixed by that single swap.

I dont want the public to come away with the erroneous idea that we can change any piece of DNA to any other piece of DNA in any human or any animal or even any cell in a dish, says Liu. But even being where we are now comes with a lot of responsibility. The big question is how much more capable will this age get? And how quickly will we be able to translate these technological advances into benefits for society?”

Crispr evolved in bacteria as a primitive defense mechanism. Its job? To find enemy viral DNA and cut it up until there was none left. Its all accelerator, no brake, and that can make it dangerous, especially for clinical applications. The longer Crispr stays in a cell, the more chances it has to find something that sort of looks like its target gene and make a cut.

To minimize these off-target effects, scientists have been developing a number of new tools to more tightly control Crispr activity.

So far, researchers have identified 21 unique families of naturally occurring anti-Crispr proteinssmall molecules that turn off the gene-editor. But they only know how a handful of them work. Some bind directly to Cas9, preventing it from attaching to DNA. Others turn on enzymes that outjostle Cas9 for space on the genome. Right now, researchers at UC Berkeley, UCSF, Harvard, the Broad, and the University of Toronto are hard at work figuring out how to turn these natural off-switches into programmable toggles.

Beyond medical applications, these will be crucial for the continued development of gene drivesa gene-editing technology that quickly spreads a desired modification through a population. Being able to nudge evolution one way or the other would be a powerful tool for combating everything from disease to climate change. Theyre being considered for wiping out malaria-causing mosquitoes, and eradicating harmful invasive species. But out in the wild, they have the potential to spread out of control, with perhaps dire consequences. Just this year Darpa poured $65 million toward finding safer gene drive designs, including anti-Crispr off-switches.

Despite decades of advances, theres still so much scientists dont understand about how bugs in your DNA can cause human disease. Even if they know what genes are coded into a cells marching orders, its a lot harder to know where those orders get delivered, and how they get translated (or mistranslated) along the way. Which is why groups at Harvard and the Broad led by Crispr co-discoverer Feng Zhang are working with a new class of Cas enzymes that target RNA instead of DNA.

Since those are the instructions that a cells machinery reads to build proteins, they carry more information about the genetic underpinnings of specific diseases. And because RNA comes and goes, making changes to it would be useful for treating short-term problems like acute inflammation or wounds. The system, which theyre calling Repair, for RNA Editing for Programmable A to I Replacement, so far only works for one nucleotide conversion. The next step is to figure out how to do the other 11 possible combinations.

And scientists are finding new Cas enzymes all the time. Teams at the Broad have also been working to characterize cpf1a version of Cas that leaves sticky ends instead of blunt ones when it cuts DNA. In February, a group from UC Berkeley discovered CasY and CasX, the most compact Crispr systems yet. And researchers expect to turn up many more in the coming months and years.

Only time will tell if Crispr-Cas9 was the best of these, or merely the first that captured the imagination of a generation of scientists. We dont know whats going to wind up working best for different applications, says Megan Hochstrasser, who did her PhD in Crispr co-discoverer Jennifer Doudnas lab and now works at the Innovative Genomics Institute. So for now I think it makes sense for everyone to be pushing on all these tools all at once.

It will take many more years of work for this generation of gene-editors to find their way out of the lab into human patients, rows of vegetables, and disease-carrying pests. That is, if gene-editing 3.0 doesnt make them all obsolete first.

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Crispr Isnt Enough Any More. Get Ready for Gene Editing 2.0

With CRISPR, geneticists have a powerful new weapon in the …

For many people today, amyotrophic lateral sclerosis, aka ALS or Lou Gehrigs disease, is most commonly linked with both the fundraising Ice Bucket Challenge and one its most famous patients, the physicist Stephen Hawking. However, it could soon have a brand-new distinction the next disease to be treatable using CRISPR-Cas9 gene-editing technology.

In work carried out by researchers at University of California, Berkeley, scientists have been able to disable the defective genethat triggers ALS in mice. While they didnt get rid of the disease permanently, the treatment did extend the mices life span by 25 percent. The therapy delayed the onset of the muscle-wasting symptoms that characterize ALS, which ultimately become fatal when they spread to the muscles which control breathing.

Some diseases, like Lou Gehrigs disease, are caused by gene mutations that lead a protein in our cells to malfunction, David Schaffer, a professor of chemical and biomolecular engineering and director of the Berkeley Stem Cell Center, told Digital Trends. A very promising approach is to disable or delete that mutated gene. CRISPR/Cas9 is a highly promising technology to do so, but this capability needs to be delivered to the target cells. We put together CRISPR-Cas9 with a highly promising gene delivery, based on a virus, in order to disable the disease causing gene SOD1 in an animal model of ALS.

The mice in the study were genetically engineered to exhibit a mutated human gene that is responsible for around 20 percent of all inherited forms of ALS. The team then used a specially engineered virusthat deliversa gene encoding the Cas9 protein,which in turn disabled the mutant gene responsible for ALS. The treated mice lived one month longer than the typical four-month life span of mice with ALS. An average healthy mouse lives for around two years.

Hopefully, were this to be carried over to humans, those time spans would beextended.There are challenges that remain before extending into human studies, such as using an improved virus optimized for humans, but we think there is a clear path to doing so, Schaffer said.

A paper describing the work was recently published in the journal Science Advances.

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CRISPR gene editing is coming to the clinic | Chemical …

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The CRISPR system uses a guide RNA (yellow) to direct the Cas9 enzyme (white) to a specific location in a cells DNA (blue) for cutting.

Credit: CRISPR Therapeutics

The gene editing technology CRISPR is one step closer to treating genetic diseases in humans. Last week, Crispr Therapeutics filed an application with European regulatory authorities to begin clinical trials for its CRISPR therapy CTX001 in a genetic blood condition called beta-thalassemia. The biotech firm expects to begin the trialthe first industry-sponsored study of a CRISPR drugnext year.

Samarth Kulkarni, CRISPR Therapeutics CEO

It is a momentous occasion for both our company and the field, says Samarth Kulkarni, CEO of Crispr Therapeutics. In early 2018, the firm will also ask the U.S. Food & Drug Administration for permission to use CTX001 to treat sickle cell disease. Vertex Pharmaceuticals today announced a 50-50 partnership with CRISPR Therapeutics to develop the drug in both beta-thalassemia and sickle cell.

Promising preclinical data presented at the American Society of Hematology (ASH) meeting in Atlanta this past weekend offered a glimpse of the strategies that Crispr Therapeutics and its many competitors are taking to treat the genetic blood diseases.

Since its conception in 2012, CRISPR has been swiftly adopted in research labs. By 2014, three biotech firms, each partly founded by one of the three inventors of CRISPR, had launched to transform the tool into a therapy. These three companies are now gearing up for their first-in-human studies, a breakneck pace for drug development.

CRISPR requires at least two basic components to edit genes: a guide RNA, which carries the code that specifies where to edit a genome, and an enzyme called Cas, which follows the guide RNA to make a cut in a cells DNA. Sometimes this cut is enough for a potential therapy. But to change the DNA or insert a new sequence, a third component, a template DNA, is required.

Both sickle cell disease and beta-thalassemia are caused by mutations in a gene that makes part of hemoglobin, the protein that carries oxygen throughout the blood. In sickle cell, the mutation causes normally donut-shaped red blood cells to warp into a crescent shape; the cells get stuck inside blood vessels, depriving tissues of oxygen. Beta-thalassemia is caused by mutations that prevent the production of fully functional hemoglobin, which for some people can cause severe anemia.

Instead of trying to fix the faulty DNA, Crispr Therapeutics and other companies are using the technology to reactivate a kind of hemoglobin found in infants. Everyone is born with high levels of a protein called fetal hemoglobin, which is mostly replaced with adult hemoglobin by three months of agethe same time that symptoms of sickle cell and beta-thalassemia appear. A gene called BCL11A represses the fetal hemoglobin production, but a rare genetic mutation in this gene is known to allow fetal hemoglobin production to continue.

Crispr Therapeutics lead drug candidate, CTX001, works by simply cutting BCL11A, basically removing the brakes on fetal hemoglobin production, Kulkarni says. On Sunday, the company presented results showing that its method edited over 90% of blood stem cells removed from patients with beta-thalassemia, dramatically increasing fetal hemoglobin in these cells.

Kulkarni says that extensive computer prediction, followed by cell and animal testing, has led them to a guide RNA in the CRISPR therapy that has no detectable off-target activitya potential side effect in which CRISPR accidently cuts the DNA in the wrong location. Stuart Orkin, a hematologist-oncologist at the Boston Childrens Hospital, says that off-target activity is one of the biggest safety concerns for gene editing in humans.

Crispr Therapeutics isnt the only company trying to increase fetal hemoglobin by targeting BCL11A. Intellia Therapeutics and Novartis have a partnership to do this with CRISPR. And Sangamo Therapeutics and Bioverativ are developing a similar therapy together using a different gene editing technology called zinc finger nucleases.

Meanwhile, Editas Medicine, another CRISPR company, presented an update at ASH on using its proprietary Cas enzyme, called Cpf1, to fix the mutation in the gene for adult hemoglobin. Charles Albright, chief scientific officer of Editas, says the company is simultaneously working on the fetal hemoglobin approach, but did not provide a timeline for when the sickle cell therapy would make it into clinical studies.

The hematology meeting also showcased many firms working on therapies for sickle cell and beta-thalassemia that dont require gene editing. Bluebird Bio, a gene therapy company, has ongoing clinical trials for both conditions using a virus to deliver a healthy copy of the hemoglobin gene into cells. Drug companies are also trying to treat sickle cell with small molecule drugs. Global Blood Therapeutics compound voxelotor, which increases hemoglobins ability to bind oxygen, is in Phase II and III clinical trials currently. And Epizyme is developing an inhibitor of an enzyme called histone methyltransferase, which could be another way to release the brakes on fetal hemoglobin production.

Everyone is working on these diseases because we know exactly what to do, and there are multiple different ways to get to the same end, a treatment, Orkin says. We dont know yet which program will be the bestBut the first one that is shown to be very effective and safe, could crowd out the others.

As it prepares to launch its first trial, Crispr Therapeutics has secured a contract manufacturer in Europe which will receive patient blood cells, edit them, and then ship them back to the clinical trial sites. Patients will then undergo chemotherapy or irradiation in preparation for their edited blood stem cells to be transplanted into the bone marrow, where they will hopefully produce healthier blood cells for life.

It is important that they do this very carefully, Orkin says. Because if there is a mistake or bad effect [from CRISPR], it will have repercussions beyond a single patient.

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