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Will CRISPR Technology Create a New "Human" Species?

How This Couple Turned Teens' Love of Texting Into Love for Reading Books

US author T.C. Boyle presents his new book 'The Women' at the Leipzig Book Fair on March 13, 2009. (Photo by JENS SCHLUETER/AFP/Getty Images)

T.C. Boyle will read his short story "Are We Not Men?" at the Los Angeles Hope Festival on Sunday, May 21. The event is free but seats are limited. RSVP here.

American author TC Boyle, who has aptly been described as "a punk Mephistopheles," talks casually about death and suicide. His interview with Big Think begins, "There is no hope whatsoever. Our species will be extinguished probably in a couple of generations, maybe even before that depending upon the microbes of the world." Yet Boyle exhibits robust mental health, maintaining an orderly writing schedulefour to five hours per day, always in the morningand a stable life, both with respect to his family and his career as a Professor of English at the University of Southern California.

Describing his short story "Are We Not Men?" Boyle says:

tc-boyle-on-writing-and-the-human-animal

"It's about CRISPR technology, which obsesses me. This is a gene editing technology which makes it much easier to edit genes in other species. In fact, if you subscribe to Nature and Science as I do for the past year there's a huge ad right in the beginning of a boxing glove on a fist and it says knock out any gene. They're selling kits to amateurs to anybody to play with various bacteria and gene edit these bacteria. Is this a good idea? I don't think so. And of course, in my telling we're just projecting slightly into the future, when we can make new species. Not to mention the parent who wants to get his kid into the best school. Give me a break. I mean it will be like buying a new car when you have a kid. You go you see how the genes line up and you pick whoever you want. You want eight foot tall? You want orange eyes? You want somebody who can run the hundred-yard dash in nine seconds? That's what it's coming to. So we're not going to be humans anymore, which I guess is no great loss."

Born Thomas John Boyle, TC changed his middle name toCoraghessan at the age of 17. As a writer, he matured at the Iowa Writer's Workshop in the 1970s, staying on after earning his MFA to complete aPhd in 19th century British Literature. While in Iowa, he forged a friendship with Raymond Carver, the best short story writer of a generation, although their two writing styles were dissimilar.

Boyle released his 26th book, The Terranauts, in October of 2016. Below is the full schedule for the Los Angeles Hope Festival.

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Will CRISPR Technology Create a New "Human" Species? - Big Think

Intellia Therapeutics has taken another step towardhuman trials of its gene editing technology after reporting new data in animal models.

The CRISPR specialist says it has been able to show for the first time that it is able to not only achieve long-term suppression of a gene using its gene-editing CRISPR/Cas9 drug, in this case the sequence coding for serum transthyretin (TTR) protein, but also demonstrate a dose-dependent reduction in the activity of the target gene in a second animal species.

New data from studies in mice show that a previously reported 97% reduction in TTRdriven by 70% gene editing efficiency working in mouse liverslasts for up to six months from a single dose. Meanwhile, a study in rats showed a similar dose-dependent reduction in the TTR gene, and crucially evidence of comparable activity in a second species as Intellia builds the case to move to the clinic.

In this test, a single intravenous infusion resulted in 66% gene editing in the rat liver and up to 91% reduction in serum TTR protein levels. Crucially, the results also backed up earlier data showing the CRISPR drug is rapidly cleared from the body, desirable as it reduces the chances of off-target effects that could cause toxicity. Both datasets were reported at the American Society of Gene & Cell Therapys Annual Meeting (ASGCT) in Washington D.C. over the weekend.

Senior VP David Morrissey said the rat study "validates the in vivo CRISPR/Cas9 platform using Intellia's proprietary LNP delivery system," adding that it shows "the ability to expand out studies in larger species.

Under FDA rules, companies typically need data in at least two animal speciesincluding one non-rodent speciesbefore they can progress into human studies. The new data keeps Intellia on course to complete the work needed to get FDA approval for trials of its TTR therapeutic in early 2018.

Companies like Intellia, Editas and CRISPR Therapeutics are vying to bring the CRISPR technology into the clinic, potentially generating one-dose therapies that could cure a host of gene-related diseases, although they will not be the first groups to do so.

Last year, Chinese scientists from Sichuan University's West China Hospital made history when they used CRISPR for the first time on an adult with lung cancer, using cells harvested from the patient that had been genetically modified using CRISPR to remove a brake (PD-1) on the immune response to the tumor. And at the end of April, a second Chinese team from Nanjing University's Nanjing Drum Tower Hospital tested a similar procedure in a patient with head and neck cancer.

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Intellia moves closer to clinic with CRISPR tech - FierceBiotech

A new website called the Pink Chicken Project offers up an intriguing nugget of an idea: what if we turned all chickens on Earth pink? Yes, you read that right. The creators of the project told Motherboard in an email that they are a small group of designers and engineers with an interest in biotechnology, and say they want to genetically modify chicken DNA so future domestic birds will be born with pink bones and pink feathers. Right now, though, the project appears to be little more than an artistic concept (complete with some photos of neon pink chicken meat, eggs, and bones).

The modification would supposedly be done using the gene editing technique CRISPR, with adoption of the pink color accelerated by a gene drive, a mechanism for increasing the odds an offspring will inherit a traitsuch as the color pinkfrom its parents. The pink color would come from cochineals, a little bug commonly used in food dye. The bug produces a chemical called carminic acid, which combines with calcium in bones to form a dye.

Why would anyone want to do this? According to the projects website, we should leave reminders for future generation of humanitys impact on the environmentin the form of discarded pink chicken bones.

The GLP aggregated and excerpted this blog/article to reflect the diversity of news, opinion, and analysis. Read full, original post:These People Want to Genetically Engineer Pink Chickens

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Coming age of CRISPR gene editing: What in heck is the 'Pink Chicken Project'? - Genetic Literacy Project

May 13, 2017 08:40 ET | Source: Intellia Therapeutics, Inc.

WASHINGTON, May 13, 2017 (GLOBE NEWSWIRE) -- Intellia Therapeutics, Inc. (NASDAQ:NTLA), a leading genome editing company focused on the development of potentially curative therapeutics using CRISPR technology, presented an update on its long-term mouse genome editing and delivery studies and shared new, first-time data in rat models demonstrating consistent dose-dependent editing, at the American Society of Gene & Cell Therapys Annual Meeting (ASGCT).

These data, featured in a platform presentation on Saturday, May 13 at ASGCT showed:

Data from the additional rat study further validates the in vivo CRISPR/Cas9 platform using Intellias proprietary LNP delivery system, said David Morrissey, Ph.D., senior vice president, Platform and Delivery Technology. In both species, we saw unprecedented in vivo liver editing results and consistent delivery of CRISPR/Cas9 with systemic administration using LNPs, while also showing the ability to expand our studies in larger species.

About Intellia Therapeutics

Intellia Therapeutics is a leading genome editing company focused on the development of proprietary, potentially curative therapeutics using the CRISPR/Cas9 system. Intellia believes the CRISPR/Cas9 technology has the potential to transform medicine by permanently editing disease-associated genes in the human body with a single treatment course. Our combination of deep scientific, technical and clinical development experience, along with our leading intellectual property portfolio, puts us in a unique position to unlock broad therapeutic applications of the CRISPR/Cas9 technology and create a new class of therapeutic products. Learn more about Intellia Therapeutics and CRISPR/Cas9 at intelliatx.com; Follow us on Twitter @intelliatweets.

Forward-Looking Statements

This press release contains "forward-looking statements" of Intellia within the meaning of the Private Securities Litigation Reform Act of 1995. These forward-looking statements include, but are not limited to, express or implied statements regarding Intellias ability to advance and expand the CRISPR/Cas9 technology to develop into human therapeutic products; our ability to achieve stable liver editing; effective genome editing with a single treatment dose; and the potential timing and advancement of our preclinical studies and clinical trials. Any forward-looking statements in this press release are based on managements current expectations and beliefs of future events, and are subject to a number of risks and uncertainties that could cause actual results to differ materially and adversely from those set forth in or implied by such forward-looking statements. These risks and uncertainties include, but are not limited to: risks related to Intellias ability to protect and maintain our intellectual property position; risks related to the ability of our licensors to protect and maintain their intellectual property position; uncertainties related to the initiation and conduct of studies and other development requirements for our product candidates; the risk that any one or more of Intellias product candidates will not be successfully developed and commercialized; the risk that the results of preclinical studies will be predictive of future results in connection with future studies; and the risk that Intellias collaborations with Novartis or Regeneron will not continue or will not be successful. For a discussion of these and other risks and uncertainties, and other important factors, any of which could cause Intellias actual results to differ from those contained in the forward-looking statements, see the section entitled Risk Factors in Intellias most recent annual report on Form 10-K filed with the Securities and Exchange Commission, as well as discussions of potential risks, uncertainties, and other important factors in Intellias subsequent filings with the Securities and Exchange Commission. All information in this press release is as of the date of the release, and Intellia Therapeutics undertakes no duty to update this information unless required by law.

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Intellia Therapeutics Announces Progress with CRISPR/Cas9 at the American Society of Gene & Cell Therapy Annual ... - GlobeNewswire (press...

New CRISPR enzymes have been found that could be used as sensitive detectors for infectious viruses.

US scientists were able to show that once CRISPR-Cas13a binds to its target RNA, it indiscriminately cuts up all RNA, like Pac-Man eating dots. Jennifer Doudna, professor of molecular biology and of chemistry at the Broad Institute, in Massachusetts, said: Our intention is to develop the Cas13a family of enzymes for point-of-care diagnostics that are robust and simple to deploy.

Researchers at the Institute paired CRISPR-Cas13a with RNA amplificationand showed the system, dubbed SHERLOCK, could detect viral RNA at extremely low concentrations. This included RNA linked to a reporter molecule that would fluoresce, allowing it to be detected. The system has been shown to detect the presence of dengue and Zika viral RNA and potentially could detect RNA of distinctive cancer cells.

The CRISPR-Cas13a family, formerly referred to as CRISPR-C2c2, is related to CRISPR-Cas9, which is already revolutionising biomedical research and treatment because of the ease of targeting it to unique DNA sequences to cut or edit. While the Cas9 protein cuts double-stranded DNA at specific sequences, the Cas13a protein a nucleic acid-cutting enzyme referred to as a nuclease latches onto specific RNA sequences, and not only cuts that specific RNA, but runs amok to cut and destroy all RNA present.

Alexandra East-Seletsky, a UC Berkeley graduate student working in the laboratory of Jennifer Doudna, one of the inventors of the CRISPR-Cas9 gene-editing tool, said: We have taken our foundational research a step further. We found other homologs of the Cas13a family that have different nucleotide preferences, enabling concurrent detection of different reporters with, say, a red and a green fluorescent signal, allowing a multiplexed enzymatic detection system.

While the original Cas13a enzyme used by the University of California Berkeley and Broad teams cuts RNA at one specific nucleic acid, uracil, three of the new Cas13a variants cut RNA at adenine. This difference allows simultaneous detection of two different RNA molecules, which could be from two different viruses.A full report of their findings will appear in Molecular Cell.

East-Seletsky said: Think of binding between Cas13a and its RNA target as an on-off switch target binding turns on the enzyme to go be a Pac-Man in the cell, chewing up all RNA nearby. This RNA killing spree can kill the cell.

UC Berkeley researchers in Nature last September argued the Pac-Man activity of CRISPR-Cas13a is its main role in bacteria, aimed at killing infectious viruses or phages. As part of the immune system of some bacteria, it allows infected cells to commit suicide to save their sister microbes from infection. Similar non-CRISPR suicide systems exist in other bacteria.

The UC Berkeley researchers subsequently searched databases of bacterial genomes and found 10 other Cas13a-like proteins. These have been synthesised and studied to assess their ability to find and cut RNA. Of those, seven resembled the original Cas13a, while three differed in where they cut RNA.

East-Seletsky said: Building on our original work, we now show that it is possible to multiplex these enzymes together, extending the scope of the technology. There is so much diversity within the CRISPR-Cas13a family that can be utilised for many applications, including RNA detection.

By Dermot Martin

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Pac-Man like CRISPR enzymes discovered - Lab News

by Tim Beck at Drexel University College of Medicine

Humanitys unnerving cruelty is perhaps only balanced by its kindness and innovation. It remains to be seen on which side of the scale CRISPR, a remarkable genome-editing tool and one of the most exciting scientific innovations of the 21st century,will land.

In their monumental 1953 Nature paper stretching over little more than one glorious page and including only a simple diagrammatic illustration and a fuzzy x-ray image Nobel laureates James Dewey Watson and Francis Harry Compton Crick proclaimed the following, We wish to suggest a structure for the salt of deoxyribose nucleic acid (D.N.A.). This structure has novel features which are of considerable biological interest. Of considerable biological interest, indeed, and a critical aspect of the discovery of CRISPR.

The Human Genome Project (HGP) taught us how to read long stretches of Watson and Cricks miraculous DNA double-helix: it taught us how to read the code of life. CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9) is the most recent step along the path towards absolute control over the human genome. CRISPR provides us with the ability to edit the code of life seemingly at will.

In the most basic of terms, CRISPR can recognize specific DNA sequences, using complementary guide RNA and recruit cutting-enzymes (e.g., the endonuclease Cas9), which precisely cut-out the targeted piece of genetic material. Under the right conditions, the cut region is subsequently filled-in with new DNA by the cells normal DNA-repair machinery.

The scientists to unlock the full genomic-editing potential of CRISPR (expanding on the work of numerous other great scientists), and likely future Nobel-laureates, Jennifer Doudna (Berkeley, California), Emmanuelle Charpentier (Max Planck Institute for Infection Biology, Berlin) and Feng Zhang (Broad MIT), published two papers in quick succession in 2012/13, describing the remarkable capability of CRISPR to precisely and reliably (to varying degrees) edit the genomes of cells, including of mammalian cells.

The promise of CRISPR technology is highlighted by the contentious ongoing patent dispute between Berkley and Broad, to clarify who discovered what when and who holds the rights to technology estimated to be worth billions of dollars.

Why is this technology worth billions of dollars? For one, think about every genetic disease you have ever heard of. Did cystic fibrosis (CF) come to mind? Do you know anyone with Tay-Sachs Disease? Duchenne Muscular Dystrophy, going once, going twice, sold to the fastest gene-editor. In theory, by combining in vitro fertilization with CRISPR technology, every known hereditary single-gene disease (you may have noticed that the above examples are all generally caused by single gene mutations) can be eliminated. Not in a year from now. Not tomorrow. Today!

Chinese researchers have validated that CRISPR/Cas9 can be used to alter human embryos, and recently Kang et al. (The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, China) used CRISPR to cure human embryos of beta-thalassemia and glucose-6-phosphate-dehydrogenase (G6PD) deficiency. The success rate (i.e., the percent of embryos that were successfully altered using CRISPR) varied significantly between studies, a concerning aspect of the technology that is likely going to be optimized rapidly, considering that CRISPR has only been in the scientific mainstream for a few short years.

Eradicating more complex diseases in embryos is a bit more complicated and will take some time. Editing genomes to treat cancer, for example, in mature individuals will also take a bit more time. However, the FDA approved a clinical trial in 2016 to test the potential of CRISPR as a tool to enhance the ability of immune cells modified outside the body, similar to the idea of modifying embryos in vitro to destroy cancer cells.

There are ethical challenges to consider. Luminaries like David Baltimore, Paul Berg and others have described these challenges in detail and have proposed appropriate steps to consider. Changes to the genome are of course permanent and as such are potentially passed on from generation to generation (i.e., changes made to an embryo are potentially passed on to subsequent offspring). Evolution at the speed of light if you will. Would it be possible to use CRISPR to create super-humans? To implant kill-switches into human cells? To prolong the lives of those that can pay for it?

The decision in February of 2016 by the UK Human Fertilisation and Embryology Authority (HFEA) to grant permission to UK researchers to edit the genome of human embryos is emblematic of the global embrace of CRISPR. Even in the US, a country generally less accommodating when it comes to research on human embryos, an international committee convened by the U.S. National Academy of Sciences (NAS) and the National Academy of Medicine concluded earlier this year that editing the DNA of a human embryo to prevent disease could be ethically permissible under the right set of circumstances.

If nothing else, history (Robert Oppenheimers opinion on the matter would be intriguing) has taught us that world-altering technologies like CRISPR cannot be un-invented. We all have front row seats to watch in awe as CRISPR transforms medicine, science and perhaps human evolution itself. Our best hope is to educate each other, to stay informed and to try to minimize the abuse of a tool powerful enough to re-write the code of life and the future of humanity.

Medical Student Editor

Drexel University College of Medicine

I am an MD/PhD Candidate at Drexel University College of Medicine/Fox Chase Cancer Center. My research focuses on cancer cell signaling, drug resistance, cancer cell invasion and discovery of prognostic biomarkers. Politics (national and international), foreign affairs and healthcare policy are additional topics I am particularly interested in.

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CRISPR: The Future of Medicine and Human Evolution - in-Training

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The researchers at the University of Pittsburgh have just used the CRISPR-Cas9 genome editing system to forever revolutionize the fight against cancer.

The treatmentwhen used on micewas shown to shrink aggressive tumors and increase survival rates without harming healthy cells. Meaning only cancer cells are attacked, effectively leaving healthy cells unharmed.

CRISPR Cancer Treatment Explained The CRISPR cancer treatment targets fusion genes, which are mutations created when two genes combine to form one hybridoften leading to cancer.

Previously, researchers found MAN2A1-FER, a fusion gene known to be associated with prostate, liver, lung, and ovarian cancer. It also contributes to thegrowth and spread of these tumors.

The unique DNA fingerprint of fusion genes could, however, be its own downfall with the CRISPR cancer treatment targeting specific DNA sequences. The treatment seeks out fusion gene patterns and replaces them with cancer-killing ones.

This is the first time that gene editing has been used to specifically target cancer fusion genes, saidJian-Hua Luo, lead author of the study. Luo added:

The tool lays the groundwork for what could become a totally new approach to treating cancer. Other types of cancer treatments target the foot soldiers of the army. Our approach is to target the command center, so there is no chance for the enemys soldiers to regroup in the battlefield for a comeback.

To test the technique, Luos team transplanted human liver and prostate cancer cells into mice and treated one group with the CRISPR cancer tool to target those fusion genes. The second group was given the same treatment targeting fusion genes that they didnt carry.

From the first group, the mices tumors shrunk up to 30 percent, didnt spread to throughout the body, and all the mice survived the eight-week test.

Meanwhile, the second group had the mices tumors grow nearly 40 times larger, spreading to other parts of the body in most cases. None of the mice in this group made it to the end of the test period.Big Plans For CRISPR-Cas9 The genome editing system has already proven itself to be an incredible tool, giving us new and amazing ways to battle muscular dystrophy, blindness, and HIV.

By also editing human immune cells to more efficiently battle cancer cells, the CRISPR cancer treatment has now been used in human trials.

It truly is an exciting time in the world of medical research as developments continue showing that the CRISPR cancer technique can remit cancer cells.

Despite this, researchers have bigger plans for the CRISPR cancer technique. They plan on testing further in hopes of completely eradicating cancer.

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What You Need to Know About the New CRISPR Cancer Treatment - BOSS Magazine

Oxford Genetics has licensed CRISPR gene editing technology from ERS Genomics. The agreement gives the British synthetic biology service provider the right to use CRISPR technology to improve gene therapy viral vectors and develop cell lines.

Oxford, United Kingdom-based Oxford Genetics has secured the nonexclusive rights to the CRISPR intellectual property. Oxford Genetics plans to use the technology to provide genome engineering services and support its cell line development and gene therapy viral vector R&D efforts. The agreement also clears Oxford Genetics to use the CRISPR-edited cells lines in the production of biotherapeutics. And to use CRISPR to develop research tools and reagents for sale.

News of the agreement comes almost exactly three years after Horizon Discovery licensed CRISPR intellectual property from ERS Genomics for use in similar applications. The nonexclusive deal between ERS Genomics and Horizon Discoverywhich is based 70 miles away from Oxford Genetics in Cambridgegave the genomics research business the right to use CRISPR to develop research tools, kits and reagents and in other applications.

ERS Genomics was cofounded by Emmanuelle Charpentier, Ph.D., one of the key players in the story of the discovery of the CRISPR-Cas9 immune system and its role in cleaving DNA. Charpentier set up the organization to facilitate access to the CRISPR-Cas9 intellectual property she holds. The firm is on the same side of the CRISPR patent dispute as CRISPR Therapeutics, Intellia Therapeutics and Caribou Biosciences. Together, the companies are appealing the U.S. patent boards ruling in the Broad Institute case.

The ruling looked at the question of whether the it was obvious to apply CRISPR to eukaryotic cells, such as the CHO and HEK293 cell lines Oxford Genetics uses in its cell line development services. But the uncertainty created by the ongoing patent dispute has not stopped Oxford Genetics from striking a deal to add CRISPR to its arsenal.

Licensing the CRISPR gene editing technology from ERS Genomics is another step on our journey to establishing the most efficient and integrated service portfolio in this sector. We are excited to be adding this technology to our existing portfolio in the synthetic biology space and supporting the rapidly expanding market for products and services that utilise genome engineering technologies, Paul Brooks, Ph.D., chief commercial officer at Oxford Genetics, said in a statement.

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Oxford Genetics licenses CRISPR tech to power synbio push - FierceBiotech

Boston Business Journal

Cambridge gene editing firm CRISPR to use delivery tech honed ... Boston Business Journal Cambridge gene editing biotech CRISPR Therapeutics has grabbed an exclusive license from MIT to use a delivery system that could make such treatments ... -$0.51 Earnings Per Share Expected for Crispr Therapeutics AG (CRSP) This QuarterThe Cerbat Gem Crispr Therapeutics AG (CRSP) Given Average Recommendation of "" by AnalystsTranscript Daily Rodger Novak Sells 49384 Shares of Crispr Therapeutics AG (CRSP) StockSports Perspectives Chaffey Breeze -Key Gazette -Petro Global News 24 all 12 news articles »

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Cambridge gene editing firm CRISPR to use delivery tech honed ... - Boston Business Journal

It's been almost two years since we learned about CRISPR, a ninja-assassin-meets-DNA-editing-tool that has been billed as one of the most powerful, and potentially controversial, technologies ever discovered by scientists. In this episode, we catch up on what's been happening (it's a lot), and learn about CRISPR's potential to not only change human evolution, but every organism on the entire planet.

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.

This episode was reported and produced by Molly Webster and Soren Wheeler. Special thanks to Jacob S. Sherkow.

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Update: CRISPR - Radiolab

Researchers have used CRISPR-Cas9 to target DNA sequences specific to cancer, shrinking tumors and improving the survival rates of cancer-stricken mice (Credit: vchalup2/Depositphotos)

The CRISPR-Cas9 genome editing system can do some pretty amazing things, giving us new ways to fight muscular dystrophy, blindness, and even HIV. But at the top of its hit list is cancer, and now researchers from the University of Pittsburgh have used the tool to target what they call cancer's command center, in a treatment that's been shown in mice to shrink aggressive tumors and increase survival rates without harming healthy cells.

The technique works by targeting fusion genes, mutations created when two separate genes combine into one hybrid that often leads to cancer. In previous work, the team found that a fusion gene known as MAN2A1-FER was associated with cancer of the prostate, liver, lungs and ovaries, and it helps the tumors grow and spread.

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But the unique DNA fingerprint of fusion genes could be their own undoing. CRISPR-Cas9 is used to target specific DNA sequences and replace them with something else, so delivering the gene editing tool through viruses, the researchers were able to seek out these fusion gene patterns and replace them with cancer-killing genes instead. The other upside is that, unlike conventional treatments like chemotherapy, the new approach will only attack cancer cells, leaving healthy cells undamaged.

In the University of Pittsburgh study, the team transplanted human prostate and liver cancer cells into mice, then treated one group with the CRISPR tool that targets those fusion genes. As a result, the tumors shrunk by up to 30 percent, didn't spread through the body, and the animals all survived to the end of the eight-week test. Meanwhile, in a control group that received the same treatment targeting fusion genes that weren't present in their bodies, the tumors grew almost 40 times larger and in most cases, spread to other parts of the body. None of the control group survived to the end of the test period.

CRISPR-Cas9 has already been put to work in human trials, but these involved editing human immune cells to better fight cancer. The new technique goes over the heads of the "foot soldiers" of the battle and instead targets the "command center" directly.

"This is the first time that gene editing has been used to specifically target cancer fusion genes," says Jian-Hua Luo, lead author of the study. " It is really exciting because it lays the groundwork for what could become a totally new approach to treating cancer. Other types of cancer treatments target the foot soldiers of the army. Our approach is to target the command center, so there is no chance for the enemy's soldiers to regroup in the battlefield for a comeback."

While the current work shows that the technique can cause the cancer cells to go into remission, the researchers plan to test whether it could be used to completely wipe it out instead.

The research was published in the journal Nature Biotechnology.

Source: University of Pittsburgh

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CRISPR gene-editing tool targets cancer's "command center" - Gizmag - New Atlas

In a study recently published online on Nature Biotechnology, University of Pittsburgh School of Medicine researchers report that a new technique using the CRISPR-Cas9 genome editing technology effectively targets cancer-causing fusion genes and improves survival in mouse models of aggressive liver and prostate cancers.

Professor of pathology at Pittsburghs School of Medicine, Jian-Hua Luo, M.D., Ph.D., explains that this is the first time that gene editing has been used to specifically target cancer fusion genes. The professor also adds that this is really exciting because it paves the way for what could become an entirely new approach to cancer treatment.

Fusion genes are hybrid genes formed from two previously separate genes. This fusion can occur as a result of gene translocation, interstitial deletion, or chromosomal inversion, and produce abnormal proteins that can cause or accelerate cancer growth. In short, these fusion genes are often associated with cancer.

A panel of fusion genes responsible for recurrent and aggressive prostate cancer has been previously identified by Dr. Luo and his team. Earlier this year, in a study published on Gastroenterology, they described how one of these fusion genes, known as MAN2A1-FER, can be found in other types of cancer, such as that of the lungs, ovaries, liver, and is also responsible for rapid tumor growth and invasiveness.

As described in a press release, for this study, the team used CRISPR-Cas9 to target unique DNA sequences formed as a result of fusion genes. The process involves the use of viruses to deliver gene editing tools that remove the mutated DNA of the fusion gene, then replacing it with genes that cause cancer cells to die.

Because fusion genes are only present in cancer cells and not healthy cells, the gene therapy is quite specific. In contrast with present cancer treatments such as chemotherapy which indiscriminately attacks both cancerous and healthy cells, this new approach will be much more preferable because it only attacks cancerous cells and leaves healthy cells intact.

To conduct the study, the team used mouse models which received transplants of human prostate and liver cancer cells. This group of mice was treated with the CRISPR gene editing tool. After the 8-week treatment period, their tumors shrunk by up to 30%, did not spread throughout their bodies, and all of them survived.

On the other hand, in a control group which was treated with viruses that target a kind of fusion gene that wasnt present in tumors, the results were a stark contrast. The mice had tumors growing almost forty times bigger. And in most cases, the tumors spread to other parts of their bodies. Most importantly, none among the group survived.

The findings suggest a new and different way to attack cancer. As Dr. Luo explained, Other types of cancer treatments target the foot soldiers of the army. Our approach is to target the command center, so there is no chance for the enemys soldiers to regroup in the battlefield for a comeback.

Dr. Luo also notes that another advantage of their approach over existing cancer treatments is that it is highly adaptive, not to mention target-specific. Going forward, they plan to test whether their strategy can do more than just stop or delay the spread of the disease. The aim of course, is to hopefully one day completely eradicate it.

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New CRISPR Technique Can Potentially Stop Cancer In Its Tracks - Wall Street Pit

Speaking of Research

A cancer gene also grows stem cells, CRISPR in monkey embryo ... Speaking of Research Welcome to this week's Research Roundup. These Friday posts aim to inform our readers about the many stories that relate to animal research each week. How To Beat Cancer? CRISPR Stares Into Its Eyes Then Snips Out ...Tech Times Genetic Engineering: We Can, But Should We? - Veritas NewsVeritas News all 3 news articles »

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A cancer gene also grows stem cells, CRISPR in monkey embryo ... - Speaking of Research

A diagnostic test for detecting Zika with CRISPR. Image: Wyss Institute

The gene editing tool CRISPR could one day mean that we can simply edit away disease, blight and undesirable genetic traits. Now, its also gaining traction in another realm of medical technology: diagnosing disease.

On Thursday, researchers at UC Berkeley announced that theyve discovered ten new CRISPR enzymes that can potentially be used to diagnose diseases like Zika or dengue fever quickly and cheaply. The technology isnt ready for prime-time yet, but it could eventually allow clinics to test a sample of someones blood, saliva, or urine for many diseases at once.

Typically, when people talk about CRISPR, they are actually talking about CRISPR-Cas9. Thats the CRISPR programming paired with one specific enzyme (Cas9) thats used to DNA at precise locations. But there are a host of other enzymes out there that can be used as part of the CRISPR system, and all of them have different talents. The new enzymes that Berkeley researchers have discovered are all variants of the CRISPR protein Cas13a, and their speciality seems to be detecting specific sequences of RNA, including those from a virus.

In genetic engineering, CRISPR is used to home in precisely on a specific piece of DNA, cut it, and put it back together with the desired genetic code. Here, the same principle is at work. Only instead of sending CRISPR to sniff out a specific piece of DNA, it hunts for RNAthe carbon copy of DNA used to make proteinsassociated with a specific virus present in someones blood, urine, saliva or other bodily fluid contains. And if CRISPR detects the genetic markers of a pathogen, it can let researchers know by fluorescing. No glow means no virus.

This method would be fantastic for cheap, point-of-care initial testing, said Alexandra East-Seletsky, the lead author on the study in Molecular Cell and a post-doc in the Berkeley lab of Jennifer Doudna, one of the scientists who initially discovered CRISPR. The power of the system is flexibility and speed for targeting new sequences, making it ideal for use during an infectious disease outbreak, or other systems requiring fast development.

The work piggy backs off earlier work by both Berkeley and the Broad Institute. In September, Berkeley researchers reported the discovery of Cas13a and its ability to detected specific sequences of RNA. Last month, the Broad Institute reported that it had used Cas13a to develop a diagnostic tool that could detect Zika and other viruses. At the time, they said that their technique was not only small and portable, but could cost as little as 61 cents per test in the field. Such a tool might detect viral and bacterial diseases, as well as potentially cancer-causing mutations.

The new work essentially expands the tools available in the toolbox, allowing the CRISPR systems to detect more than one thing at once.

It allows you to test a control substrate and an unknown at the same time, or look for multiple disease-related sequences at once using the same starting material, said East-Seletsky. The possibilities are endless.

Developing cheap, bedside disease detection is a sort of Holy Grail of medicine. CRISPRs potential role here has received considerably less attention than its ability to edit genes, but it could wind up being equally significant.

Still, East-Seletsky said, Thursdays study is just a proof of concept. There is yet a lot of work to be done before there is actually a CRISPR-based diagnostic tool available to clinics.

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CRISPR Could Transform the Way We Diagnose Disease - Gizmodo

TechCrunch

Scientists have eliminated HIV in mice using CRISPR TechCrunch An important breakthrough has been made in the eradication of AIDs. Scientists have found they can successfully snip out the HIV virus from mouse cells using CRISPR/Cas9 technology. Right now patients with the deadly virus must use a toxic concoction ... CRISPR and the Dawn of the New Biotech RevolutionReason (blog) Researchers use gene editing to eliminate HIV infection in miceCBS News Closer to a cure: CRISPR cuts HIV from its cellular hideoutNew Atlas Genetic Engineering & Biotechnology News -Geek -ScienceDirect -Temple Health all 85 news articles »

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Scientists have eliminated HIV in mice using CRISPR - TechCrunch

May 04, 2017 08:00 ET | Source: CRISPR Therapeutics AG

BASEL, Switzerland and CAMBRIDGE, Mass., May 04, 2017 (GLOBE NEWSWIRE) -- CRISPR Therapeutics (NASDAQ:CRSP), a biopharmaceutical company focused on developing transformative gene-based therapeutics for patients with serious diseases, has promoted Dr. Samarth Kulkarni, Ph.D. to the role of President and Chief Business Officer of CRISPR Therapeutics Inc., as announced today by Dr. Rodger Novak, M.D., Chief Executive Officer of CRISPR Therapeutics.The new role reflects Dr. Kulkarnis increased responsibilities in leading the strategic direction of the company and overseeing its U.S. operations.Dr. Kulkarni will continue to lead strategy, business development, investor relations and external communications in his expanded role.

Over the past two years as Chief Business Officer, Dr. Kulkarni had a leading role in the establishment of its key collaborations with Vertex and Bayer, and played a major part in helping finance the companys operations through its IPO. Sam has played a pivotal role in enabling the rapid growth of the company, and we look forward to his continued leadership, said Dr. Rodger Novak. Additionally, as we rapidly move our lead programs to the clinic, we will look to further expand our senior management with leaders having deep expertise in later-stage clinical development and registration of breakthrough therapies.

CRISPR Therapeuticss lead program, which aims to provide a functional cure for beta thalassemia and sickle cell disease, is on track and the company is planning to file for a clinical trial authorization in Europe by the end of 2017. CRISPR Therapeutics Inc., is a wholly-owned subsidiary and base of R&D operations for CRISPR Therapeutics AG, parent company of the CRISPR group.

About CRISPR Therapeutics

CRISPR Therapeutics is a leading gene-editing company focused on developing transformative gene-based medicines for serious diseases using its proprietary CRISPR / Cas9 gene-editing platform. CRISPR / Cas9 is a revolutionary technology that allows for precise, directed changes to genomic DNA. The company's multi-disciplinary team of world-class researchers and drug developers is working to translate this technology into breakthrough human therapeutics in a number of serious diseases. Additionally, CRISPR Therapeutics has established strategic collaborations with Bayer AG and Vertex Pharmaceuticals to develop CRISPR-based therapeutics in diseases with high unmet need. The foundational CRISPR / Cas9 patent estate for human therapeutic use was licensed from the company's scientific founder Emmanuelle Charpentier, Ph.D. CRISPR Therapeutics AG is headquartered in Basel, Switzerland, with its wholly-owned U.S. subsidiary, CRISPR Therapeutics, Inc., and R&D operations based in Cambridge, Massachusetts. For more information, please visit http://www.crisprtx.com.

CRISPR Forward-Looking Statement

Certain statements set forth in this press release constitute forward-looking statements within the meaning of the Private Securities Litigation Reform Act of 1995, as amended, including, but not limited to, statements concerning: the therapeutic value, development and the commercial potential of CRISPR/Cas-9 gene editing technologies.. You are cautioned that forward-looking statements are inherently uncertain. Although the company believes that such statements are based on reasonable assumptions within the bounds of its knowledge of its business and operations, the forward-looking statements are neither promises nor guarantees and they are necessarily subject to a high degree of uncertainty and risk. Actual performance and results may differ materially from those projected or suggested in the forward-looking statements due to various risks and uncertainties. These risks and uncertainties include, among others: uncertainties inherent in the initiation and conduct of preclinical and clinical studies for the companys product candidates; availability and timing of results from preclinical and clinical studies; whether results from a preclinical study or clinical trial will be predictive of future results in connection with future trials or use; expectations for regulatory approvals to conduct trials or to market products; and those risks and uncertainties described in Item 1A under the heading Risk Factors in the companys annual report on Form 10-K, and in any other subsequent filings made by the company with the U.S. Securities and Exchange Commission (SEC), which are available on the SECs website at https://www.sec.gov. Existing and prospective investors are cautioned not to place undue reliance on these forward-looking statements, which speak only as of the date they are made. The information contained in this press release is provided by the company as of the date hereof, and, except as required by law, the company disclaims any intention or responsibility for updating or revising any forward-looking information contained in this press release.

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CRISPR Therapeutics Appoints Samarth Kulkarni, Ph.D. as President, Expanding Role Beyond Chief Business Officer ... - GlobeNewswire (press release)

The European Centre for Disease Prevention and Control (ECDC) released more information today about the do-it-yourself CRISPR (Cas 9) kits found to be contaminated with harmful bacteria, saying the risk of infection is low but does exist.

The kits were identified by the Bavarian Health and Food Safety Authority on Mar 24. As a result, the ECDC said Germany has halted all importation of the DIY Bacterial Gene Engineering CRISPR Kit, manufactured in the United States and sold on the Internet for $150.

The gene-editing kit is labeled as containing a harmless laboratory strain of Escherichia coli HME63, but tests on two kits ordered from the United States in November of 2016 and in March showed contamination with several pathogenic bacteria, including antibiotic-resistant bacteria.

The ECDC identified the pathogens as Klebsiella pneumoniae, Enterobacter, and Enterococcus faecalis, which belong to biological risk group 2. Biological risk group 2 pathogens require safety handling, including wearing personal protective equipment.

Despite the presence of risk group 2 pathogens, the ECDC identified the risk of infection for users of the kits as low "because the manipulation of the kit does not involve percutaneous injury-prone manipulations. However, infection resulting from the contamination of broken skin or mucous membranes may occur, even though the kit recommends and provides disposable gloves."

At-home CRISPR kits are targeted at hobbyists and "citizen" scientists who want to participate in at-home experiments with genetic engineering by making precision genome edits in bacteria. The kits have become popular in recent years, and The Odin, the company selling the kit identified by German health officials, currently has a waiting list of about a week.

Though the risk of infection is low, the ECDC said that the bacteria from the kits could invade a human gastrointestinal track. "Bacteria with resistance can persist for several months in the intestinal tract of asymptomatic carriers. If a carrier develops severe illness and requires antimicrobial treatment, there is a potential risk that the antibiotic-resistant bacteria proliferate and subsequently cause multidrug-resistant infection."

Finally, the ECDC said that users of the CRISPR kit should dispose of their material in a safe way, so as not to introduce the multidrug resistant-bacteria into the environment. The agency didn't specify steps for safe disposal.

See also:

May 3 ECDC risk assessment

May 3 ECDC news release

CRISPR kit Web site

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ECDC says risk from contaminated CRISPR kits low - CIDRAP

In 2012, Cal biochemistry and molecular biology professor Jennifer Doudna and microbiologist Emmanuelle Charpentier, now of the Max Planck Institute, changed the world. They invented CRISPR-Cas9, a gene editing tool

Then in 2013, MIT bioengineer Feng Zhang published a paper in the journal Science that outlined a CRISPR process specifically for eukaryotic cells, i.e., those from higher plants and animalsAt that point, the CRISPR saga bifurcated into two parts: the research narrative and the legal fight.

[Berkeley law professor Robert ]Merges set it up this way: UC maintains that it has never been determined who first developed eukaryotic CRISPR applications, that CRISPR basically uses the same process for viruses and eukaryotic cells, and that the February decision should be reversed. But the patent trial court found that there is no interference in fact, which in this case basically means the inventions are not the same, said Merges.

Its like Cal claims it invented cookies, and then Broad says it invented chocolate-chip cookies, he says. If Cals [pending] patent is verified and Broads also is upheld, you could end up with a situation where a biotech company would need licenses from both Cal and Broad for a CRISPR application. That kind of bundled license is very common in the world ofpatents.

The GLP aggregated and excerpted this blog/article to reflect the diversity of news, opinion, and analysis. Read full, original post: The Current CRISPR Patent Dispute, Explained

For more background on the Genetic Literacy Project, read GLP on Wikipedia

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What you need to know about the legal battle over CRISPR patents - Genetic Literacy Project

With the advent of CRISPR , a new way to edit DNA, the field of genomic technology has never been more exciting. The implications have yet to be seen, but scientists could theoretically snip out a person's genetic risk for disease. But it's also never been a more anxiety-inducing time. Some experts argue innovations in genomics are moving forward at a pace faster than our ability to parse their potential consequences.

In a panel discussion at Fortune s Brainstorm Health conference in San Diego, scientists discussed the promises and perils of this breakthrough technologysome of which they're already starting to see.

I think CRISPR is a very exciting discovery, said J. Craig Venter, co-founder of the health company Human Longevity, Inc. and one of the first scientists to sequence the human genome. Venter is using genome sequencing as a way to help predict a persons risk for disease and offer more personalized treatment with a physical exam called the Health Nucleus : an eight-hour, $25,000 inside-and-out doctors appointment that includes whole-genome sequencing, high-tech scanning and early diagnostics.

So far the company has sequenced more than 40,000 human genomes. Of the people that complete the Health Nucleus, one in 40 will discover they have a serious cancer they didn't know about, he said.

Yet some experts are skeptical that exhaustive testing always translates to better health. Dr. Eric Topol, founder and director of Scripps Translational Science Institute, called for a more reserved way forward in his remarks at the conference, arguing that too much scanning can lead to more false positive results and potentially unnecessary interventions. We have to prove that doing tests are truly associated with positive outcomes, Topol said. We have to be much more discriminative about the tests that we do.

Some companies are taking a more tempered approach: inexpensive testing that looks for specific genes known to substantially increase a persons risk for disease. Color Genomics, a genetics company that has brought down the cost of genetic testing, focuses on cancer and offers affordable tests for the BRCA1 and BRCA2 genes, which can significantly raise a womans risk for breast and ovarian cancer. It changes the equation in terms of treating disease, said Othman Laraki, co-founder and CEO of Color Genomics.

As for finding and fixing genetic problems well before they even arise? The scientists on the panel agreed that they're not there yet, and that current iterations of CRISPR may not be quite as precise as the hype has claimed. For now, that may be for the best. Editing human embryos with CRISPR should be a long way off, said Venter. Not something we do next week.

More here:

How Scientists Think CRISPR Will Change Medicine - TIME

Brian Nolan is a partner in the Life Sciences Group of Mayer Brown LLP.

The European Patent Office (EPO) and the United States Patent and Trademark Office (USPTO) have been thrust into a dispute pitting the University of California, Berkeley (UC) and the University of Vienna against the Broad Institute of MIT and Harvard University. The patent applications in the dispute disclose the genome editing tool called CRISPR/Cas9. Scientists and inventors have been watching the decisions of the EPO and USPTO because these decisions may leave one of these entities with a patent portfolio worth billions of dollars.

The Broad Institute scored the first victory at the USPTO, which held that the Institute is entitled to claims for the use of CRISPR-Cas9 in eukaryotic cells because that use is patentably distinct from UC's earlier disclosure showing the ability to use CRISPR-Cas9 in vitro. But just a few weeks later, the EPO informed the public that it intends to issue a patent to UC for the use of CRISPR-Cas9 in either prokaryotic or eukaryotic cells.

The CRISPR-Cas9 genome editing system finds its scientific roots in the observation that prokaryotes utilise CRISPR to identify and destroy viruses. Professor Jennifer Doudna of UC and Professor Emmanuelle Charpentier of the University of Vienna showed that the CRISPR-Cas9 system could use short strands of guide RNA that recognize and direct Cas9 to locate a specific target DNA sequence, and to cut it. In four preliminary patent applications filed between May 2012 and February 2013 and a related scientific paper published on 17 August, 2012, Professor Doudna and her colleagues Dr Martin Jinek of UC and Professor Charpentier disclosed a successful test of a prokaryotic CRISPR-Cas9 genome editing tool in vitro.

Around the same time, Dr Feng Zhang, a scientist at the Broad Institute, was working on creating and using various bacterial Cas9 in mammalian cells. Between December 2012 and June 2013, a few months after Professor Doudna and Dr Charpentier's publication, Professor Zhang and his colleagues filed several provisional patent applications showing the use of CRISPR-Cas9 with human and mouse cells. Professor Zhang and his colleagues requested and paid for expedited review. The expedited review resulted in US patents being issued to Professor Zhang and colleagues before the issuance of patents to Professor Doudna and her colleagues.

UC and the Broad Institute have been watching the progression of each other's patent applications through the USPTO and EPO. When UC saw that the USPTO had issued patents to the Institute for the use of CRISPR-Cas9 in eukaryotic cells, UC requested that the USPTO decide whether UC's patent disclosure showing the use of CRISPR-Cas9 genome editing system in vitro in prokaryotic cells evidenced that using CRISPR-Cas9 in eukaryotic cells was UC's invention. The USPTO instituted a proceeding called an 'interference' to review the parties' claims.

During this proceeding, UC alleged that Zhang's work was an obvious extension of that shown in Professor Doudna's article. Based upon an email from a former member of Professor Zhang's laboratory, Shuailiang Lin, to Professor Doudna, UC asserted that Professor Zhang began only meaningfully working on CRISPR-Cas9 after reading that article. The Institute responded by characterizing the email as that of a former employee seeking a new job and willing to tell a prospective employer what they wanted to hear.

In response to this, the Broad Institute responded that the USPTO should not have instituted this proceeding because its patent claims are distinct from UC's patent applications, which only show success of CRISPR-Cas9 in vitro. In February, the USPTO ruled in the Broad Institute's favour and held that 'the parties claim patentably distinct subject matter, rebutting the presumption created by declaration of this interference. [The] Broad [Institute] provided sufficient evidence to show that its claims, which are all limited to CRISPR-Cas9 systems in a eukaryotic environment, are not drawn to the same invention as UC's claims, which are all directed to CRISPR-Cas9 systems not restricted to any environment.'

The USPTO came to this conclusion because it determined that the evidence showed that a person of ordinary skill in the art would not have a reasonable expectation that CRISPR-Cas9 could be implemented successfully in a eukaryotic cell based upon the success UC achieved in vitro. To support its conclusion, the USPTO pointed to several statements by Professor Doudna and Dr Jinek, the UC inventors, that questioned whether the CRISPR-Cas9 genome editing system could be implemented in a eukaryotic environment. UC has appealed the USPTO's decision to a United States appellate court. Yet as the appellate court will make its decision on the same record presented to the USPTO, it may be difficult for UC to convince the appellate court that substantial evidence did not support the USPTO's determination. The appeal should be resolved with 10-12 months.

In parallel with the USPTO's review, the EPO was considering what, if any, patent protection it should grant UC. UC's European patent application was the subject of great interest, as several third-party observations were submitted to the EPO seeking to impede the issuance of the patent. These observations included arguments that the UC patent publications did not support a claim scope that encompassed CRISPR in a eukaryotic environment, similar to those presented in the United States. Despite these submissions, the EPO has notified the public that it intends to issue a patent to UC for the use of CRISPR-Cas9 in either prokaryotic or eukaryotic cells. Within nine months of issuance, this patent may be the subject of third-party oppositions that challenge whether the patent should have issued. If filed, an opposition will afford the third-party a substantive role in the arguments before the EPO than it had before, similar to the procedure in the US where a third-party cab remain involved during an interference proceeding. This may increase the likelihood of the EPO questioning a claim scope that includes use in a eukaryotic cell.

Either way, the EPO will be unlikely to resolve an opposition until late 2018 or 2019; so as with the patent situation in the United States, the dispute will continue in Europe. Unless the parties enter into a settlement, the industry should expect uncertainty with respect to ownership of the use of CRISPR-Cas9 in eukaryotic cells for the next two to four years, with the potential for various disputes to erupt in the patent offices and courts of the United States and Europe.

The decisions of the USPTO and the EPO to grant rights to the use of CRISPR-Cas9 systems in a eukaryotic cell to different parties has complicated commercialisation decisions for biopharma companies. It is thought that the more lucrative uses for CRISPR-Cas9 will be in a eukaryotic environment. Thus, if the status quo remains, throughout the duration of the patents that may last until 2033, companies will have to secure licensing rights from both UC and the Broad Institute if they plan on commercialising products in both the United States and Europe which is the likely plan for biopharma companies. Therefore, biopharma companies will continue to watch these CRISPR-Cas9 patent disputes until either the disputes are resolved - or a significantly different CRISPR system becomes the genome editing tool of choice.

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The CRISPR patent dispute - Europe and the US - BioNews

"During acute infection, HIV actively replicates," explained co-senior study investigator Kamel Khalili, Ph.D., professor and chair of the department of neuroscience at LKSOM. "With EcoHIV mice, we were able to investigate the ability of the CRISPR/Cas9 strategy to block viral replication and potentially prevent systemic infection." The excision efficiency of their strategy reached 96% in EcoHIV mice, providing the first evidence for HIV-1 eradication by prophylactic treatment with a CRISPR/Cas9 system.

In the third animal model, a latent HIV-1 infection was recapitulated in humanized mice engrafted with human immune cells, including T cells, followed by HIV-1 infection. "These animals carry latent HIV in the genomes of human T cells, where the virus can escape detection, Dr. Hu explained. Amazingly, after a single treatment with CRISPR/Cas9, viral fragments were successfully excised from latently infected human cells embedded in mouse tissues and organs.

In all three animal models, the researchers employed a recombinant adeno-associated viral (rAAV) vector delivery system based on a subtype known as AAV-DJ/8. "The AAV-DJ/8 subtype combines multiple serotypes, giving us a broader range of cell targets for the delivery of our CRISPR/Cas9 system," remarked Dr. Hu. Additionally, the researchers re-engineered their previous gene-editing apparatus to now carry a set of four guide RNAs, all designed to efficiently excise integrated HIV-1 DNA from the host cell genome and avoid potential HIV-1 mutational escape.

To determine the success of the strategy, the team measured levels of HIV-1 RNA and used a novel and cleverly designed live bioluminescence imaging system. "The imaging system, developed by Dr. Won-Bin Young while at the University of Pittsburgh, pinpoints the spatial and temporal location of HIV-1-infected cells in the body, allowing us to observe HIV-1 replication in real time and to essentially see HIV-1 reservoirs in latently infected cells and tissues," stated Dr. Khalili.

The researchers were excited by their findings and are optimistic about their next steps. The next stage would be to repeat the study in primates, a more suitable animal model where HIV infection induces disease, in order to further demonstrate the elimination of HIV-1 DNA in latently infected T cells and other sanctuary sites for HIV-1, including brain cells," Dr. Khalili concluded. "Our eventual goal is a clinical trial in human patients."

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CRISPR Eliminates HIV in Live Animals - Genetic Engineering & Biotechnology News

Using CRISPR to editthe fusion genes that can cause or worsen cancer reduced the size of tumors and improved survival in mice, report researchers.

This is the first time that gene editing has been used to specifically target cancer fusion genes. It is really exciting because it lays the groundwork for what could become a totally new approach to treating cancer, explains lead study author Jian-Hua Luo, professor of pathology at University of Pittsburghs School of Medicine and director of its High Throughput Genome Center.

Fusion genes, which often are associated with cancer, form when two previously separate genes become joined together and produce an abnormal protein that can cause or promote cancer.

Luo and his team had previously identified a panel of fusion genes responsible for recurrent and aggressive prostate cancer. In a study published earlier this year in the journal Gastroenterology, the team reported that one of these fusion genes, known as MAN2A1-FER, also is found in several other types of cancer, including that of the liver, lungs, and ovaries, and is responsible for rapid tumor growth and invasiveness.

In the current study, published online in Nature Biotechnology, the researchers employed the CRISPR-Cas9 genome editing technology to target unique DNA sequences formed because of the gene fusion. The team used viruses to deliver the gene editing tools that cut out the mutated DNA of the fusion gene and replaced it with a gene that leads to death of the cancer cells.

Because the fusion gene is present only in cancer cells, not healthy ones, the gene therapy is highly specific. Such an approach could come with significantly fewer side effects when translated to the clinic, which is a major concern with other cancer treatments such as chemotherapy.

To conduct the study, the researchers used mouse models that had received transplants of human prostate and liver cancer cells. Editing the cancer fusion gene resulted in up to 30 percent reduction in tumor size. None of the mice exhibited metastasis and all survived during the eight-week observation period.

In contrast, in control mice treated with viruses designed to cut out another fusion gene not present in their tumors, the tumors increased nearly 40-fold in size, metastasis was observed in most animals, and all died before the end of the study.

The new findings suggest a completely new way to combat cancer. Other types of cancer treatments target the foot soldiers of the army. Our approach is to target the command center, so there is no chance for the enemys soldiers to regroup in the battlefield for a comeback, says Luo.

Another advantage over traditional cancer treatment is that the new approach is very adaptive. A common problem that renders standard chemotherapies ineffective is that the cancer cells evolve to generate new mutations. Using genome editing, the new mutations could be targeted to continue fighting the disease, Luo notes.

In the future, the researchers plan to test whether this strategy could completely eradicate the disease rather than induce the partial remission observed in the current study.

Grants from the National Institutes of Health, the Department of Defense, and the University of Pittsburgh Cancer Institute supported this work.

Source: University of Pittsburgh

Link:

Using CRISPR against cancer shows success in mice - Futurity - Futurity: Research News

They found that the protein kinase called Polo-like kinase 4 (PLK4), which is increased in AT/RT, regulates these tumors growth, survival and tendency to metastasize.

Interestingly, Sredni had performed a number of genomics studies previously, and this specific marker had never popped up before.

If I hadnt done this functional study, where, knocking out the PLK4 gene, resulted in the inhibition of cell proliferation I wouldnt have discovered that PLK4 is essential for AT/RT growth. PLK4 is only slightly elevated in these tumors. However, this is gene is tightly regulated and slight increases in its expression results in an aggressive tumor phenotype. This overexpression can be targeted by inhibitors opening a new therapeutic prospective for children with AT/RT. Significantly, we also found it elevated in other embryonal tumors of the brain, what may have a larger impact in patient care, Sredni said. The fact that we used CRISPR for the functional assay was key.

Even more promising, Sredni and her team were able to target the kinase with an available PLK4 inhibitor currently being tested for breast cancer that so far is providing promising results.

Srednis group experiments showed that the drug significantly impaired AT/RT cell proliferation, survival, migration and invasion.

More interestingly, it appears that the drug does not affect normal cells. The researchers tested the toxicity of the drug in normal human fibroblasts and in zebrafish larvae, exposing them to high doses of the drug for extended periods of time and found that it did not affect the fibroblasts and did not increase death or affected development of zebrafish larvae. This is a sign that the drug may be safe for use in pediatrics.

The team has now moved on to studies in mice with intracranial xenograft. While this is still under development, Sredni said it looks like tumors are actually growing significantly slower or even shrinking.

So Im knocking on wood here and hoping for good news soon she said.

CRISPR made a big impact on this research and Sredni, like many, think the tools reach will be broad.

The advent of CRISPR I think its going to change science from now on, in every aspect, she said. Its really user-friendly, robust and reliable.

Of course its not perfect, it still needs some optimization, Sredni acknowledged. But, it is powerful! For example in my hands, with a small team and the help of my summer students, we were able to mutate individual kinases, observe the cells phenotype and make significant new discoveries.

While she thinks progress in therapeutics will be more challenging than discovery, she added, CRISPR will really make a difference in what we will be doing from now on in cancer research.

Next, Sredni will perform high-throughput analyses on her mutated and treated cells in order to find downstream targets that will be adequate for combined therapies.

One drug alone wont cure cancer, she said.

The team is working on developing data for an application for federal funding in order to fully characterize the phenomenon observed so far. If all goes well, Sredni sees a Phase 1 clinical trial coming up soon.

The findings were published in Pediatric Blood & Cancer.

The rest is here:

Using CRISPR to Find Treatments for Aggressive Pediatric Brain Cancer - Bioscience Technology

BY ALICE PARK

Dr. Carl Junes lab at the University of Pennsylvania looks like any other biology research hub. There are tidy rows of black-topped workbenches flanked by shelves bearing boxes of pipettes and test tubes. Theres ad hoc signage marking the different workstations. And there are postdocs buzzing around, calibrating scales, checking incubators and smearing solutions and samples onto small glass slides.

Appearances aside, what June is attempting to do here, on the eighth floor of the glass-encased Smilow Center for Translational Research in Philadelphia, is anything but ordinary. Hes built a career trying to improve the odds for people with intractable end-stage disease, and now, in the universitys brand-new cell-processing lab, hes preparing to launch his most ambitious study yet: hes going to try to treat 18 people with stubborn cancers, and hes going to do it using CRISPR, the most controversial new tool in medicine.

Developed just four short years ago by two groupsJennifer Doudna, a molecular and cell biologist at the University of California, Berkeley, together with Emmanuelle Charpentier, now at the Max Planck Institute in Berlin; and Feng Zhang, a biomedical engineer at the Broad Institute of Harvard and MITCRISPR allows scientists to easily and inexpensively find and alter virtually any piece of DNA in any species. In 2016 alone it was used to edit the genes of vegetables, sheep, mosquitoes and all kinds of cell samples in labs. Now, even as some scientists call for patience and extreme caution, theres a worldwide race to push the limits of CRISPRs capabilities.

Junes ultimate goal is to test CRISPRs greatest potential: its ability to treat diseases in humans. Before we were kind of flying in the dark when we were making gene changes, he says of earlier attempts at genetic tinkering. With CRISPR, I came to the conclusion that this technology needs to be tested in humans. The trial, which will start treating patients in a few months, is the first to use this powerful technique in this way. It represents the most extensive manipulation of the human genome ever attempted.

Soon, Junes 18 trial patients will become the first people in the world to be treated with CRISPRd cellsin this case, cells genetically edited to fight cancer. Like many people with cancer, the patients have run out of options. So, building on work by Doudna, Charpentier and Zhang, Junes team will extract their T cells, a kind of immune cell, and use CRISPR to alter three genes in those cells, essentially transforming them into superfighters. The patients will then be reinfused with the cancer-fighting T cells to see if they do what theyre supposed to do: seek and destroy cancerous tumors.

A lot of hope hangs on the outcome of the trial, but whether it succeeds or fails, it will provide scientists with critical information about what can go right and wrong when they try to rewrite the genetic code in humans. The hope is that studies like Junes will bear out CRISPRs therapeutic potential, leading to the development of radical new therapies not just for people with the cancers being studied but for all of them, as well as for genetic diseases such as sickle-cell anemia and cystic fibrosis, and chronic conditions like Type 2 diabetes and Alzheimers. It may sound far-fetched, but studies like this one are an enormous first step in that direction.

Using CRISPR on humans is still hugely controversial, in part because its so easy. The fact that it allows scientists to efficiently edit any genefor some cancers, but also potentially for a predisposition for red hair, for being overweight, for being good at mathworries ethicists because of what could happen if it gets into the wrong hands. As of now, the National Institutes of Health (NIH), by far the worlds largest sponsor of scientific research, will not fund studies using CRISPR on human embryos. And any new way of altering genes in human cells must get ethics and safety approval by the NIH, regardless of who is paying for it. (The NIH also opposes the use of CRISPR on so-called germ-line cellsthose in an egg, sperm or embryosince any such changes would be permanent and heritable.)

To fund his study, June was able to attract support from Sean Parker, the former Facebook executive and Silicon Valley entrepreneur behind Napster. Parker recently founded the $250 million Parker Institute for Cancer Immunotherapy, a collaboration among six major cancer centers, and Junes study is its first ambitious undertaking. We need to take big, ambitious bets to advance cancer treatment, says Parker. Were trying to lead the way in doing more aggressive, cutting-edge stuff that couldnt get funded if we werent around.

Thats not to say Junes study will necessarily cure these cancers. Either its back to the drawing board, he says, or everyone goes forward and studies a wide variety of other diseases that could potentially be fixed. In reality, both things are probably true.

Even if Junes study doesnt work as he hopes, experts still agree it will be a matter of monthsnot yearsbefore other privately funded human studies get launched in the U.S. and abroad. An ongoing patent battle over who owns the lucrative technology hasnt stopped investors from pouring millions into CRISPR companies. So simple and inexpensive is the technique, and so frenzied is the medical community about its potential, that it would be foolish to bet on anything else. With a technology like CRISPR, says Doudna, youve lit a fire.

A Year of Progress CRISPRs journey from lab bench to cancer treatment may seem quick. After all, as recently as a couple of years ago only a minuscule number of people even knew what clustered regularly interspaced short palindromic repeatsthats longhand for CRISPRwas. But the technology is at least hundreds of millions of years old. It was bacteria that originally used CRISPR, as a survival mechanism to fend off infection by viruses. The ultimate freeloaders, viruses never bothered developing their own reproductive system, preferring instead to insert their genetic material into that of other cellsincluding bacteria. Bacteria fought back, holding on to snippets of a virus genes when they were infected. The bacteria would then surround these viral DNA fragments with a genetic sequence that effectively cut them out altogether.

Bacteria have been performing that clever evolutionary stunt for millennia, but it wasnt until the early 2000s that food scientists at a Danish yogurt company realized just how clever the bacterial system was when they noticed that their cultures were turning too sour. They discovered that the cultures were CRISPRing invaders, altering the taste considerably. It made for bad dairy, but the scientific discovery was immediately recognized as a big one.

About a decade later, in 2012, Doudna and Charpentier tweaked the system to make it more standardized and user-friendly, and showed that not just bacterial DNA but any piece of DNA has this ability. That was a game changer. Scientists have been mucking with plant, animal and human DNA since its structure was first discovered by James Watson and Francis Crick in 1953. But altering genes, especially in deliberate, directed ways, has never been easy. The idea of gene correction is not new at all, says June. But before CRISPR it just never worked well enough so that people could do it routinely.

Within months of Doudnas and Charpentiers discovery, Zhang showed that the technique worked to cut human DNA at specified places. With that, genetics changed overnight. Now scientists had a tool allowing them, at least in theory, to wield unprecedented control over any genome, making it possible to delete bits of DNA, add snippets of genetic material and even insert entirely new pieces of code.

Now, that theoretical potential took shape in a remarkable array of real-world applications. CRISPR produced the first mushroom that doesnt brown, the first dogs with DNA-boosted cells giving them a comic-book-like musculature, and a slew of nutritionally superior crops that are already on their way to market. There are even efforts to use CRISPRd mosquitoes to fight Zika and malaria.

On the human side, progress has been even more dramatic. In a lab, scientists have successfully snipped out HIV from infected human cells and demonstrated that the process works in infected mice and rats as well. Theyre making headway in correcting the genetic defect behind sickle-cell anemia, which stands to actually cure the disease. Theyre making equally promising progress in treating rare forms of genetic blindness and muscular dystrophy. And in perhaps the most controversial application of CRISPR to date, in 2016 the U.K. approved the first use of the technology in healthy human embryos for research.

At the Francis Crick Institute in London, developmental biologist Kathy Niakan is using CRISPR to try to understand one of the more enduring mysteries of human development: what goes wrong at the earliest stages, causing an embryo to die and a pregnancy to fail. To be clear, Niakan will not attempt to implant the embryos in a human; her research is experimental, and the embryos are destroyed seven days after the studies begin.

Like Niakan, June is looking for answers to one of human biologys more vexing problems: why the immune system, designed to fight disease, is nearly useless against cancer. Its an issue thats kept him up at night since 2001, when his wife, not responding to the many treatments she tried, died of ovarian cancer.

This trial is about two things: safety and feasibility, he says. Its about testing whether its even possible to successfully edit these immune cells to make them doin human bodies, not a petri dishwhat he wants them to do. Either way, the study will yield critical information, paving the way for eventual new treatment options that are more targeted, less brutal and far smarter against tumors than systemwide chemotherapy will ever be.

As much as has been done in 2016, this is only the beginning of a kind of medicine that stands to effectively change the course of human history. CRISPR is an empowering technology with broad applications in both basic science and clinical medicine, says Dr. Francis Collins, director of the NIH. It will allow us to tackle problems that for a long time we probably felt were out of our reach.

The Hurdles Ahead Because its so easy to use, Zhang, along with the other CRISPR pioneers, says careful thought should be given to where and how it gets employed. For the most part I dont think we are getting ahead of ourselves with the CRISPR applications, he says. What we need to do is really engage the public, to make sure people understand what are the really exciting potential applications and what are the immediate limitations of the technology, so we really are applying it and supporting it in the right way.

Regulatory scrutiny is a given with CRISPR, and any new tool for rewriting human DNA requires federal approval. For the current Penn trial, June got the green light from the NIH Recombinant DNA Advisory Committee, established in the 1980s to assess the safety of any first-in-humans gene-therapy trials. While there are still dangers involved in any kind of gene therapythe changes may happen in unexpected places, for example, or the edits may have unanticipated side effectsscientists have learned more about the best way to make the genetic changes, and how to deliver them more safely. So far, animal studies show CRISPR provides enough control that unexpected negative effects are rareat least so far.

The role of regulatory oversight is less clear when the technique is used to alter food crops. Even before Junes patients get infused with CRISPRd T cells, farmers in Argentina and Minnesota will plant the worlds first gene-edited crops for market. CRISPR provides an unparalleled ability to insert almost any trait into plantsdrought or pest resistance, more of this vitamin or less of that nutritional villain du jour. Dupont, for instance, is putting the finishing touches on its first drought-resistant corn, and biotech company Calyxt has created a potato that doesnt produce cancerous compounds when fried; its also planting its first crop of soy plants modified to produce higher amounts of healthy oleic-acid fats.

These edits involve deleting or amping up existing genesnot adding new ones from other speciesand the U.S. Department of Agriculture has said this kind of gene-edited food crop is not significantly different from unaltered crops and therefore does not need to be regulated differently.

In the coming months, the National Academy of Sciences is expected to issue guidelines that might address some of the challenges posed by CRISPR, focusing on how and when to proceed with developing new disease treatments. The report is expected to launch much-needed discussion in the scientific community and among the public as well. Whether more regulation will eventually be required likely depends on how far scientists push the limits of their editingand how comfortable consumers and advocacy groups are with those studies.

As CRISPR goes mainstream in medicine and agriculture, profound moral and ethical questions will arise. Few would argue against using CRISPR to treat terminal cancer patients, but what about treating chronic diseases? Or disabilities? If sickle-cell anemia can be corrected with CRISPR, should obesity, which drives so many life-threatening illnesses? Who decides where that line ought to be drawn?

Questions like these weigh heavily on June and all of CRISPRs pioneering scientists. Having this technology enables humans to alter human evolution, says Doudna. Thinking about all the different ways it can be employed, both for good and potentially not for very good, I felt it would be irresponsible as someone involved in the earliest stages of the technology not to get out and talk about it.

Last year, Doudna invited other leaders in genetics to a summit to address the immediate concerns about applying CRISPR to human genes. The group agreed to a voluntary temporary moratorium on using CRISPR to edit the genes of human embryos that would be inserted into a woman and brought to term, since the full array of CRISPRs consequences isnt known yet. (Any current research using human embryos, including Niakans, is lab-only.)

For researchers like June and Niakan, Doudna and Zhang, and others, proceeding carefully with CRISPR is the only way forward. But proceed they will. The sooner more answers emerge, the sooner CRISPR can mature and begin to deliver on its promise. There are thousands of applications for CRISPR, says June. The sky is the limit. But we have to be careful.

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CRISPR Technology Scientists on Their Gene Editing Tool - TIME

Cas9 (CRISPR associated protein 9) is an RNA-guided DNA endonuclease associated with the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) type II adaptive immunity system in Streptococcus pyogenes, among other bacteria. S. pyogenes utilizes Cas9 to interrogate and cleave foreign DNA,[1] such as invading bacteriophage DNA or plasmid DNA.[2] Cas9 performs this interrogation by unwinding foreign DNA and checking whether it is complementary to the 20 basepair spacer region of the guide RNA. If the DNA substrate is complementary to the guide RNA, Cas9 cleaves the invading DNA. In this sense, the CRISPR-Cas9 mechanism has a number of parallels with the RNA interference (RNAi) mechanism in eukaryotes. Native Cas9 assists in all three CRISPR steps: it participates in adaptation, participates in crRNA processing and it cleaves the target DNA assisted by crRNA and an additional RNA called tracrRNA. Native Cas9 requires a guide RNA composed of two disparate RNAs that associate to make the guide - the CRISPR RNA (crRNA), and the trans-activating RNA (tracrRNA).,[3][4]

The Cas9 protein has been heavily utilized as a genome engineering tool to induce site-directed double strand breaks in DNA. These breaks can lead to gene inactivation or the introduction of heterologous genes through non-homologous end joining and homologous recombination respectively in many laboratory model organisms. Alongside zinc finger nucleases and TALEN proteins, Cas9 is becoming a prominent tool in the field of genome editing. Cas9 has gained traction in recent years because it can cleave nearly any sequence complementary to the guide RNA.[2] Because the target specificity of Cas9 stems from the guide RNA:DNA complementarity and not modifications to the protein itself (like TALENs and Zinc-fingers), engineering Cas9 to target new DNA is straightforward.[5][6] Versions of Cas9 that bind but do not cleave cognate DNA can be used to localize transcriptional activator or repressors to specific DNA sequences in order to control transcriptional activation and repression.[7][8] Cas9 targeting has been simplified through the engineering of a chimeric single guide RNA. Scientists have suggested that Cas9-based gene drives may be capable of editing the genomes of entire populations of organisms.[9] In 2015, scientists in China used Cas9 to modify the genome of human embryos for the first time.[10]

To survive in a variety of challenging, inhospitable habitats that are filled with bacteriophage, bacteria have evolved methods to evade and fend off predatory viruses. This includes the recently appreciated CRISPR system. CRISPR loci are composed of short, palindromic repeats that occur at regular intervals composed of alternate CRISPR repeats and variable CRISPR spacers. These CRISPR loci are usually accompanied by adjacent CRISPR-associated (cas) genes. In 2005, it was discovered by three separate groups that the spacer regions were homologous to foreign DNA elements, including plasmids and viruses. These reports provided the first biological evidence that CRISPRs might function as an immune system.

CRISPR-Cas systems are divided into two classes and many subtypes, based on their genetic content and structural differences. However, the core defining features of all CRISPR-Cas systems are the cas genes and their proteins: cas1 and cas2 are universal while cas3, cas9, and cas10 are signature genes specific subtypes.

The CRISPR-Cas defense can be described in three stages:

The three stages of the CRISPR-Cas adaptive immune system, based on the CRISPR-Cas system in Streptococcus thermophilus.

Stage 1: CRISPR spacer integration. Protospacers and protospacer-associated motifs (shown in red) are acquired at the leader end of a CRISPR array in the host DNA. The CRISPR array is composed of spacer sequences (shown in colored boxes) flanked by repeats (black diamonds). This process requires Cas1 and Cas2 (and Cas9 in type II[1]), which are encoded in the cas locus, which are usually located near the CRISPR array.

Stage 2: CRISPR expression. Pre-crRNA is transcribed starting at the leader region by the host RNA polymerase and then cleaved by Cas proteins into smaller crRNAs containing a single spacer and a partial repeat (shown as hairpin structure with colored spacers).

Stage 3: CRISPR interference. crRNA with a spacer that has strong complementarity to the incoming foreign DNA begins a cleavage event (depicted with scissors), which requires Cas proteins. DNA cleavage interferes with viral replication and provides immunity to the host. The interference stage can be functionally and temporarily distinct from CRISPR acquisition and expression (depicted by white line dividing the cell) [4].

Cas9 features a bi-lobed architecture with the guide RNA nestled between the alpha-helical lobe (blue) and the nuclease lobe (cyan, orange and gray). These two lobes are connected through a single bridge helix. There are two nuclease domains located in the multi-domain nuclease lobe, the RuvC (gray) which cleaves the non-target DNA strand, and the HNH nuclease domain (cyan) that cleaves the target strand of DNA. Interestingly, the RuvC domain is encoded by sequentially disparate sites that interact in the tertiary structure to form the RuvC cleavage domain.

A key feature of the target DNA is that it must contain a protospacer adjacent motif (PAM) consisting of the three-nucleotide sequence- NGG. This PAM is recognized by the PAM-interacting domain (PI domain, orange) located near the C-terminal end of Cas9. Cas9 undergoes distinct conformational changes between the apo, guide RNA bound, and guide RNA:DNA bound states, which are detailed below. PAM is recognized by Arg 1333 and Arg 1335 in the major groove by a - hairpin, where they bind to dG2 and dG3 of PAM.[14]

Cas9 recognizes the stem-loop architecture inherent in the CRISPR locus, which mediates the maturation of crRNA-tracrRNA ribonucleoprotein complex.[15] Cas9 in complex with CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) further recognizes and degrades the target dsDNA.[16] In the co-crystal structure shown here, the crRNA-tracrRNA complex is replaced by a chimeric single-guide RNA (sgRNA, in red) which has been proved to have the same function as the natural RNA complex.[17] The sgRNA base paired with target ssDNA is anchored by Cas9 as a T-shaped architecture. This crystal structure of the DNA-bound Cas9 enzyme reveals distinct conformational changes in the alpha-helical lobe with respect to the nuclease lobe, as well as the location on the HNH domain. protein consists of a recognition lobe (REC) and a nuclease lobe (NUC). It should be noted that all regions except the HNH form tight interactions with each other and sgRNA-ssDNA complex, while the HNH domain forms few contacts with the rest of the protein. In another conformation of Cas9 complex observed in the crystal, the HNH domain is not visible. These structures suggest the conformational flexibility of HNH domain.

Several crystal structures have been published, including:

In sgRNA-Cas9 complex, based on the crystal structure, REC1, BH and PI domains have important contacts with backbone or bases in both repeat and spacer region.[23][24] Several Cas9 mutants including REC1 or REC2 domains deletion and residues mutations in BH have been tested. REC1 and BH related mutants show lower or none activity compared with wild type, which indicate these two domains are crucial for the sgRNA recognition at repeat sequence and stabilization of the whole complex. Although the interactions between spacer sequence and Cas9 as well as PI domain and repeat region need further studies, the co-crystal demonstrates clear interface between Cas9 and sgRNA. Indeed, the recent crystal structure of Cas9 bound to single-guide RNA reveals that the 10-nucleotide RNA seed sequence is preordered in an A-form conformation for target DNA recognition. In addition to the pre-ordered seed sequences, comparison of the Cas9-sgRNA complex with the target DNA-bound structure (PDB 4UN3) reveals that the PAM-interacting sites (R1333 and R1335) responsible for 5-NGG-3 PAM recognition are pre-positioned prior to binding target DNA. Together, these structural observations show that the spacer region of sgRNA, especially the seed region, is essential for triggering Cas9 to form a DNA recognition-competent structure that is ready to engage double-stranded DNA target sequences.[21]

Previous sequence analysis and biochemical studies have suggested Cas9 contain RNase H and HNH endonuclease homologous domains which are responsible for cleavages of two target DNA strands, respectively. These results are finally proved in the structure. Although the low sequence similarity, the sequence similar to RNase H has a RuvC fold (one member of RNase H family) and the HNH region folds as T4 Endo VII (one member of HNH endonuclease family). Previous works on Cas9 have demonstrated that HNH domain is responsible for complementary sequence cleavage of target DNA and RuvC is responsible for the non-complementary sequence (Westra, et al. 2012; Wiedenheft, et al. 2014).

Due to the unique ability of Cas9 to bind to essentially any complement sequence in any genome, researchers wanted to use this enzyme to repress transcription of various genomic loci. To accomplish this, the two crucial catalytic residues of the RuvC and HNH domain can be mutated to alanine abolishing all endonuclease activity of Cas9. The resulting protein coined dead Cas9 or dCas9 for short, can still tightly bind to dsDNA. This catalytically inactive Cas9 variant has been used for both mechanistic studies into Cas9 DNA interrogative binding and as a general programmable DNA binding RNA-Protein complex.

The interaction of dCas9 with target dsDNA is so tight that high molarity urea protein denaturant can not fully dissociate the dCas9 RNA-protein complex from dsDNA target.[25] dCas9 has been targeted with engineered single guide RNAs to transcription initiation sites of any loci where dCas9 can compete with RNA polymerase at promoters to halt transcription.[26] Also, dCas9 can be targeted to the coding region of loci such that inhibition of RNA Polymerase occurs during the elongation phase of transcription.[26] In Eukaryotes, silencing of gene expression can be extented by targeting dCas9 to enhancer sequences, where dCas9 can block assembly of transcription factors leading to silencing of specific gene expression.[8] Moreover, the guide RNAs provided to dCas9 can be designed to include specific mismatches to its complementary cognate sequence that will quantitatively weaken the interaction of dCas9 for its programmed cognate sequence allowing a researcher to tune the extent of gene silencing applied to a gene of interest.[26] This technology is similar in principle to RNAi such that gene expression is being modulated at the RNA level. However, the dCas9 approach has gained much traction as there exist less off-target effects and in general larger and more reproducible silencing effects through the use of dCas9 compared to RNAi screens.[27] Furthermore, because the dCas9 approach to gene silencing can be quantitatively controlled, a researcher can now precisely control the extent to which a gene of interest is repressed allowing more questions about gene regulation and gene stoichiometry to be answered.

Beyond direct binding of dCas9 to transcriptionally sensitive positions of loci, dCas9 can be fused to a variety of modulatory protein domains to carry out a myriad of functions. Recently, dCas9 has been fused to chromatin remodeling proteins(HDACs/HATs) to reorganize the chromatin structure around various loci.[26] This is an important in targeting various eukaryotic genes of interest as heterochromatin structures hinder Cas9 binding. Furthermore, because Cas9 can react to heterochromatin, it is theorized that this enzyme can be further applied to studying the chromatin structure of various loci.[26] Additionally, dCas9 has been employed in genome wide screens of gene repression. By employing large libraries of guide RNAs capable of targeting thousands of genes, genome wide genetic screens using dCas9 have been conducted.[28]

Another method for silencing transcription with Cas9 is to directly cleave mRNA products with the catalytically active Cas9 enzyme.[29] This approach is made possible by hybridizing ssDNA with a PAM complement sequence to ssRNA allowing for a dsDNA-RNA PAM site for Cas9 binding. This technology makes available the ability to isolate endogenous RNA transcripts in cells without the need to induce chemical modifications to RNA or RNA tagging methods.

In contrast to silencing genes, dCas9 can also be used to activate genes when fused to transcription activating factors.[26] These factors include subunits of bacterial RNA Polymerase II and traditional transcription factors in Eukaryotes. Recently, genome wide screens of transcription activation have also been accomplished using dCas9 fusions named CRISPRa for activation.[28]

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Cas9 - Wikipedia