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  • richardmitnick 10:21 am on July 3, 2017 Permalink | Reply
    Tags: , CRISPR, , , ,   

    From HMS: “Bringing CRISPR into Focus” 

    Harvard University

    Harvard University

    Harvard Medical School

    Harvard Medical School

    June 29, 2017
    KEVIN JIANG

    CRISPR-Cas3 is a subtype of the CRISPR-Cas system, a widely adopted molecular tool for precision gene editing in biomedical research. Aspects of its mechanism of action, however, particularly how it searches for its DNA targets, were unclear, and concerns about unintended off-target effects have raised questions about the safety of CRISPR-Cas for treating human diseases.

    Harvard Medical School and Cornell University scientists have now generated near-atomic resolution snapshots of CRISPR that reveal key steps in its mechanism of action. The findings, published in Cell on June 29, provide the structural data necessary for efforts to improve the efficiency and accuracy of CRISPR for biomedical applications.

    Through cryo-electron microscopy, the researchers describe for the first time the exact chain of events as the CRISPR complex loads target DNA and prepares it for cutting by the Cas3 enzyme. These structures reveal a process with multiple layers of error detection—a molecular redundancy that prevents unintended genomic damage, the researchers say.

    High-resolution details of these structures shed light on ways to ensure accuracy and avert off-target effects when using CRISPR for gene editing.

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

    Target search

    Discovered less than a decade ago, CRISPR-Cas is an adaptive defense mechanism that bacteria use to fend off viral invaders. This process involves bacteria capturing snippets of viral DNA, which are then integrated into its genome and which produce short RNA sequences known as crRNA (CRISPR RNA). These crRNA snippets are used to spot “enemy” presence.

    Acting like a barcode, crRNA is loaded onto members of the CRISPR family of enzymes, which perform the function of sentries that roam the bacteria and monitor for foreign code. If these riboprotein complexes encounter genetic material that matches its crRNA, they chop up that DNA to render it harmless. CRISPR-Cas subtypes, notably Cas9, can be programmed with synthetic RNA in order to cut genomes at precise locations, allowing researchers to edit genes with unprecedented ease.

    To better understand how CRISPR-Cas functions, Liao partnered with Ailong Ke of Cornell University. Their teams focused on type 1 CRISPR, the most common subtype in bacteria, which utilizes a riboprotein complex known as CRISPR Cascade for DNA capture and the enzyme Cas3 for cutting foreign DNA.

    Through a combination of biochemical techniques and cryo-electron microscopy, they reconstituted stable Cascade in different functional states, and further generated snapshots of Cascade as it captured and processed DNA at a resolution of up to 3.3 angstroms—or roughly three times the diameter of a carbon atom.

    1
    A sample cryo-electron microscope image of CRISPR molecules(left). The research team combined hundreds of thousands of particles into 2D averages (right), before turning them into 3D projections. Image: Xiao et al.

    Seeing is believing

    In CRISPR-Cas3, crRNA is loaded onto CRISPR Cascade, which searches for a very short DNA sequence known as PAM that indicates the presence of foreign viral DNA.

    Liao, Ke and their colleagues discovered that as Cascade detects PAM, it bends DNA at a sharp angle, forcing a small portion of the DNA to unwind. This allows an 11-nucleotide stretch of crRNA to bind with one strand of target DNA, forming a “seed bubble.”

    The seed bubble acts as a fail-safe mechanism to check whether the target DNA matches the crRNA. If they match correctly, the bubble is enlarged and the remainder of the crRNA binds with its corresponding target DNA, forming what is known as an “R-loop” structure.

    Once the R-loop is completely formed, the CRISPR Cascade complex undergoes a conformational change that locks the DNA into place. It also creates a bulge in the second, non-target strand of DNA, which is run through a separate location on the Cascade complex.

    Only when a full R-loop state is formed does the Cas3 enzyme bind and cut the DNA at the bulge created in the non-target DNA strand.

    The findings reveal an elaborate redundancy to ensure precision and avoid mistakenly chopping up the bacteria’s own DNA.

    2
    CRISPR forms a “seed bubble” state, which acts as an initial fail-safe mechanism to ensure that CRISPR RNA matches its target DNA. Image: Liao Lab/HMS

    “To apply CRISPR in human medicine, we must be sure the system is accurate and that it does not target the wrong genes,” said Ke, who is co-senior author of the study. “Our argument is that the CRISPR-Cas3 subtype has evolved to be a precise system that carries the potential to be a more accurate system to use for gene editing. If there is mistargeting, we know how to manipulate the system because we know the steps involved and where we might need to intervene.”

    Setting the sights

    Structures of CRISPR Cascade without target DNA and in its post-R-loop conformational states have been described, but this study is the first to reveal the full sequence of events from seed bubble formation to R-loop formation at high resolution.

    In contrast to the scalpel-like Cas9, CRISPR-Cas3 acts like a shredder that chews DNA up beyond repair. While CRISPR-Cas3 has, thus far, limited utility for precision gene editing, it is being developed as a tool to combat antibiotic-resistant strains of bacteria. A better understanding of its mechanisms may broaden the range of potential applications for CRISPR-Cas3.

    In addition, all CRISPR-Cas subtypes utilize some version of an R-loop formation to detect and prepare target DNA for cleavage. The improved structural understanding of this process can now enable researchers to work toward modifying multiple types of CRISPR-Cas systems to improve their accuracy and reduce the chance of off-target effects in biomedical applications.

    “Scientists hypothesized that these states existed but they were lacking the visual proof of their existence,” said co-first author Min Luo, postdoctoral fellow in the Liao lab at HMS. “The main obstacles came from stable biochemical reconstitution of these states and high-resolution structural visualization. Now, seeing really is believing.”

    “We’ve found that these steps must occur in a precise order,” Luo said. “Evolutionarily, this mechanism is very stringent and has triple redundancy, to ensure that this complex degrades only invading DNA.”

    Additional authors on the study include Yibei Xiao, Robert P. Hayes, Jonathan Kim, Sherwin Ng, and Fang Ding.

    This work is supported by National Institutes of Health

    See the full article here .

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  • richardmitnick 1:15 pm on June 25, 2017 Permalink | Reply
    Tags: , , Bacteriophages, CRISPR, Genetically modified viruses,   

    From Nature: “Modified viruses deliver death to antibiotic-resistant bacteria” 

    Nature Mag
    Nature

    21 June 2017
    Sara Reardon

    Engineered microbes turn a bacterium’s immune response against itself using CRISPR.

    1
    Phages (green) attack a bacterium (orange). Researchers are hoping to use engineered versions of these viruses to fight antibiotic resistance. AMI Images/SPL

    Genetically modified viruses that cause bacteria to kill themselves could be the next step in combating antibiotic-resistant infections [Nature].

    Several companies have engineered such viruses, called bacteriophages, to use the CRISPR gene-editing system to kill specific bacteria, according to a presentation at the CRISPR 2017 conference in Big Sky, Montana, last week. These companies could begin clinical trials of therapies as soon as next year.

    Initial tests have saved mice from antibiotic-resistant infections that would otherwise have killed them, said Rodolphe Barrangou, chief scientific officer of Locus Biosciences in Research Triangle Park, North Carolina, at the conference.

    Bacteriophages isolated and purified from the wild have long been used to treat infections in people, particularly in Eastern Europe. These viruses infect only specific species or strains of bacteria, so they have less of an impact on the human body’s natural microbial community, or microbiome, than antibiotics do. They are also generally thought to be very safe for use in people.

    But the development of phage therapy has been slow, in part because these viruses are naturally occurring and so cannot be patented. Bacteria can also quickly evolve resistance to natural phages, meaning researchers would have to constantly isolate new ones capable of defeating the same bacterial strain or species. And it would be difficult for regulatory agencies to continually approve each new treatment.

    CRISPR-fuelled death

    To avoid these issues, Locus and several other companies are developing phages that turn the bacterial immune system known as CRISPR against itself. In Locus’ phages, which target bacteria resistant to antibiotics, the CRISPR system includes DNA with instructions for modified guide RNAs that home in on part of an antibiotic-resistance gene. Once the phage infects a bacterium, the guide RNA latches on to the resistance gene. That prompts an enzyme called Cas3, which the bacterium normally produces to kill phages, to destroy that genetic sequence instead. Cas3 eventually destroys all the DNA, killing the bacterium. “I see some irony now in using phages to kill bacteria,” says Barrangou.

    Another company, Eligo Bioscience in Paris, uses a similar approach. It has removed all the genetic instructions that allow phages to replicate, and inserted DNA that encodes guide RNAs and the bacterial enzyme Cas9. Cas9 cuts the bacterium’s DNA at a designated spot, and the break triggers the bacterium to self-destruct. The system will target human gut pathogens, says Eligo chief executive Xavier Duportet, although he declined to specify which ones.

    The two companies hope to start clinical trials in 18–24 months. Their first goal is to treat bacterial infections that cause severe disease. But eventually, they want to develop phages that let them precisely engineer the human microbiome by removing naturally occurring bacteria associated with conditions such as obesity, autism and some cancers.

    Both Barrangou and Duportet acknowledge that for now, causal links between the human microbiome and these conditions are tenuous at best. But they hope that by the time their therapies have been proved safe and effective in humans, the links will be better understood. Phages could also allow researchers to manipulate the microbiomes of experimental animals, which could help them to untangle how certain bacteria influence conditions such as autism, says Timothy Lu, a synthetic biologist at the Massachusetts Institute of Technology in Cambridge and a co-founder of Eligo.

    An engineered solution

    Other companies are working to get phages to perform different tasks. ‘Supercharged’ phages, created by a group at Synthetic Genomics in La Jolla, California, could contain dozens of special features, including enzymes that break down biofilms or proteins that help to hide the phages from the human immune system.

    But engineered phages still have to overcome some hurdles. Treating an infection might require a large volume of phages, says Elizabeth Kutter, a microbiologist at Evergreen State College in Olympia, Washington, and it’s unclear whether this would trigger immune reactions, some of which could interfere with the treatment. Phages could also potentially transfer antibiotic-resistance genes to non-resistant bacteria, she notes.

    Lu adds that bacteria may still develop resistance even to the engineered phages. So researchers might have to frequently modify their phages to keep up with bacterial mutations.

    But as antibiotic resistance spreads, Kutter says, there will be plenty of space for both engineered phages and natural phage therapies, which are also growing in popularity. “I think they’ll complement the things that can be done by natural phages that have been engineered for hundreds of thousands of years,” she says.

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  • richardmitnick 5:23 pm on June 2, 2016 Permalink | Reply
    Tags: , , CRISPR, Researchers unlock new CRISPR system for targeting RNA   

    From Broad Institute: “Researchers unlock new CRISPR system for targeting RNA” 

    Broad Institute

    Broad Institute

    June 2nd, 2016
    Broad Institute of MIT and Harvard
    Paul Goldsmith
    617-714-8600
    paulg@broadinstitute.org

    Discovered in bacteria as viral defense mechanism, researchers program C2c2 to manipulate cellular RNA using CRISPR

    Researchers from the Broad Institute of MIT and Harvard, Massachusetts Institute of Technology, the National Institutes of Health, Rutgers University-New Brunswick and the Skolkovo Institute of Science and Technology have characterized a new CRISPR system that targets RNA, rather than DNA.

    The new approach has the potential to open a powerful avenue in cellular manipulation. Whereas DNA editing makes permanent changes to the genome of a cell, the CRISPR-based RNA-targeting approach may allow researchers to make temporary changes that can be adjusted up or down, and with greater specificity and functionality than existing methods for RNA interference.

    1
    A team led by Feng Zhang of the Broad and MIT and Eugene Koonin of the NIH has revealed that C2c2 helps protect bacteria against viral
    infection by targeting RNA. Photo composite by Lauren Solomon, Broad communications. Images courtesy of Broad communications and NIH.

    In a study* published today in Science, Feng Zhang and colleagues at the Broad Institute and the McGovern Institute for Brain Research at MIT, along with co-authors Eugene Koonin and his colleagues at the NIH, and Konstantin Severinov of Rutgers University-New Brunswick and Skoltech, report the identification and functional characterization of C2c2, an RNA-guided enzyme capable of targeting and degrading RNA.

    The findings reveal that C2c2—the first naturally-occurring CRISPR system that targets only RNA to have been identified, discovered by this collaborative group in October 2015—helps protect bacteria against viral infection. They demonstrate that C2c2 can be programmed to cleave particular RNA sequences in bacterial cells, which would make it an important addition to the molecular biology toolbox.

    The RNA-focused action of C2c2 complements the CRISPR-Cas9 system, which targets DNA, the genomic blueprint for cellular identity and function. The ability to target only RNA, which helps carry out the genomic instructions, offers the ability to specifically manipulate RNA in a high-throughput manner—and manipulate gene function more broadly. This has the potential to accelerate progress to understand, treat and prevent disease.

    “C2c2 opens the door to an entirely new frontier of powerful CRISPR tools,” said Feng Zhang, senior author, and Core Institute Member of the Broad Institute. “There are an immense number of possibilities for C2c2 and we are excited to develop it into a platform for life science research and medicine.”

    “The study of C2c2 uncovers a fundamentally novel biological mechanism that bacteria seem to use in their defense against viruses,” said Eugene Koonin, senior author, and leader of the Evolutionary Genomics Group at the NIH’s National Center for Biotechnology Information. “Applications of this strategy could be quite striking.”

    Currently, the most common technique for performing gene knockdown is small interfering RNA (siRNA). According to the researchers, C2c2 RNA-editing methods suggest greater specificity and hold the potential for a wider range of applications, such as:

    Adding modules to specific RNA sequences to alter their function—how they are translated into proteins—which would make them valuable tools for large-scale screens and constructing synthetic regulatory networks, and
    Harnessing C2c2 to fluorescently tag RNAs as a means to study their trafficking and subcellular localization.

    In this work, the team was able to precisely target and remove specific RNA sequences using C2c2 – lowering the expression level of the corresponding protein. This suggests C2c2 could represent an alternate approach to siRNA, complementing the specificity and simplicity of CRISPR-based DNA editing and offering researchers adjustable gene “knockdown” capability using RNA.

    C2c2 has advantages that make it suitable for tool development:

    C2c2 is a two-component system, requiring only a single guide RNA to function, and
    C2c2 is genetically encodable—meaning the necessary components can be synthesized as DNA for delivery into tissue and cells.

    “C2c2’s greatest impact may be made on our understanding the role of RNA in disease and cellular function,” said co-first author Omar Abudayyeh, a graduate student in the Zhang Lab.

    *Science paper:
    C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector

    See the full article here .

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  • richardmitnick 3:18 pm on May 3, 2016 Permalink | Reply
    Tags: , , CRISPR,   

    From AAAS: “The gene editor CRISPR won’t fully fix sick people anytime soon. Here’s why” 

    AAAS

    AAAS

    May. 3, 2016
    Jocelyn Kaiser

    1
    Researchers still have a ways to go before using CRISPR to repair genes in patients. iStock

    This week, scientists will gather in Washington, D.C., for an annual meeting devoted to gene therapy—a long-struggling field that has clawed its way back to respectability with a string of promising results in small clinical trials. Now, many believe the powerful new gene-editing technology known as CRISPR will add to gene therapy’s newfound momentum. But is CRISPR really ready for prime time? Science explores the promise—and peril—of the new technology.

    How does CRISPR work?

    Traditional gene therapy works via a relatively brute-force method of gene transfer. A harmless virus, or some other form of so-called vector, ferries a good copy of a gene into cells that can compensate for a defective gene that is causing disease. But CRISPR can fix the flawed gene directly, by snipping out bad DNA and replacing it with the correct sequence. In principle, that should work much better than adding a new gene because it eliminates the risk that a foreign gene will land in the wrong place and turn on a cancer gene. And a CRISPR-repaired gene will be under the control of that gene’s natural promoter, so the cell won’t make too much or too little of its protein product.

    What has CRISPR accomplished so far?

    Researchers have published successes with using CRISPR to treat animals with an inherited liver disease and muscular dystrophy, and there will be more such preclinical reports at this week’s annual meeting of the American Society of Gene and Cell Therapy (ASGCT). The buzz around CRISPR is growing. This year’s meeting includes 93 abstracts on CRISPR (of 768 total), compared with only 33 last year. What’s more, investors are flocking to CRISPR. Three startups, Editas Medicine, Intellia Therapeutics, and CRISPR Therapeutics, have already attracted hundreds of millions of dollars.

    So why isn’t CRISPR ready for prime time?

    CRISPR still has a long way to go before it can be used safely and effectively to repair—not just disrupt—genes in people. That is particularly true for most diseases, such as muscular dystrophy and cystic fibrosis, which require correcting genes in a living person because if the cells were first removed and repaired then put back, too few would survive. And the need to treat cells inside the body means gene editing faces many of the same delivery challenges as gene transfer—researchers must devise efficient ways to get a working CRISPR into specific tissues in a person, for example.

    CRISPR also poses its own safety risks. Most often mentioned is that the Cas9 enzyme that CRISPR uses to cleave DNA at a specific location could also make cuts where it’s not intended to, potentially causing cancer.

    With these caveats, do you even need CRISPR?

    Conventional gene addition treatments for some diseases are so far along that it may not make sense to start over with CRISPR. In Europe, where one gene therapy is already approved for use for a rare metabolic disorder, regulators are poised to approve a second for an immune disorder known as adenosine deaminase–severe combined immunodeficiency (SCID). And in the United States, a company this year expects to seek approval for a gene transfer treatment for a childhood blindness disease called Leber congenital amaurosis (LCA).

    At the ASCGT meeting, researchers working with the company Bluebird Bio will present interim data for a late-stage trial showing that gene addition can halt the progression of cerebral adrenoleukodystrophy, a devastating childhood neurological disease. Final results could help pave the way for regulatory approval. Bluebird will also report on trials using gene transfer for two blood disorders, sickle cell disease and β-thalassemia, bringing these treatments closer to the clinic.

    Except for LCA, in which gene-carrying viruses are injected directly into eyes, these diseases are treated by removing bone marrow cells from patients, adding a gene to the cells, and reinfusing the cells back into the patient. New, safer viral vectors have reduced risks of leukemia seen in a few patients in some early trials for immunodeficiency diseases. Researchers are seeing “excellent clinical responses,” says Donald Kohn of the University of California, Los Angeles.

    Although Kohn and other researchers have used an older gene-editing tool known as zinc finger nucleases to repair defective genes causing sickle cell disease and a type of SCID in cells in a dish, only a tiny fraction of immature blood cells needed for the therapy to work end up with the gene corrected—far below the fraction altered by now standard gene transfer methods. One reason is because the primitive blood cells aren’t dividing much (more on this below). Because gene-editing methods such as CRISPR are so much less efficient than gene addition, for several diseases, “I don’t think there will be a strong rationale for switching to editing,” says Luigi Naldini of the San Raffaele Telethon Institute for Gene Therapy in Milan, Italy.

    CRISPR also has other issues

    Using CRISPR to cut out part of a gene—not correct the sequence—is relatively easy to do. In fact, this strategy is already being tested with zinc finger nucleases in a clinical effort to stop HIV infection. In this treatment, the nucleases are used to knock out a gene for a receptor called CCR5 in blood cells that HIV uses to get into cells.

    But when CRISPR is used to correct a gene using a strand of DNA that scientists supply to cells, not just to snip out some DNA, it doesn’t work very well. That’s because the cells must edit the DNA using a process called homology-directed repair, or HDR, that is only active in dividing cells. And unfortunately, most cells in the body—liver, neuron, muscle, eye, blood stem cells—are not normally dividing. For this reason, “knocking out a gene is a lot simpler than knocking in a gene and correcting a mutation,” says Cynthia Dunbar, president-elect of ASGCT and a gene therapy researcher at the National Heart, Lung, and Blood Institute in Bethesda, Maryland.

    Researchers are working on ways to get around this limitation. The genes for HDR are present in all cells, and it’s a matter of turning them on, perhaps by adding certain drugs to the cells, says CRISPR researcher Feng Zhang of the Broad Institute in Cambridge, Massachusetts. Another avenue is to find alternatives to the Cas9 system that don’t rely on the HDR process, Zhang says.

    But the low rate of HDR in most cells is one reason why the first use of CRISPR in the clinic will likely involve disrupting genes, not fixing them. For example, several labs have shown in mice that CRISPR can remove a portion of the defective gene that causes Duchenne muscular dystrophy, so that the remaining portion will produce a functional, albeit truncated protein. Editas hopes to start a clinical trial next year to treat a form of LCA blindness by chopping out part of the defective gene. One proposed gene-editing treatment for sickle cell disease would similarly snip out some DNA, so that blood cells produce a fetal form of the oxygen-carrying protein hemoglobin.

    And CRISPR still has big safety risks

    The most-discussed safety risk with CRISPR is that the Cas9 enzyme, which is supposed to slice a specific DNA sequence, will also make cuts in other parts of the genome that could result in mutations that raise cancer risk. Researchers are moving quickly to make CRISPR more specific. For example, in January, one lab described a tweak to Cas9 that dramatically reduces off-target effects. And in April in Nature, another team showed how to make the enzyme more efficient at swapping out single DNA bases.

    But immediate off-target cuts aren’t the only worry. Although it’s possible to deliver CRISPR’s components into cells in a dish as proteins or RNA, so far researchers can usually only get it working in tissue inside the body by using a viral vector to deliver the DNA for Cas9 into cells. This means that even after Cas9 has made the desired cuts, cells will keep cranking it out. “The enzyme will still hang around over 10, 20 years,” Zhang says. That raises the chances that even a very specific Cas9 will still make off-target cuts and that the body will mount an immune response to the enzyme.

    This may not truly be a problem, Zhang suggests. His team created a mouse strain that is born with the gene for Cas9 turned on all the time, so it expresses the enzyme in all cells for the animal’s entire life. Even after interbreeding these mice for about 20 generations, the mice “seem to be fine” with no obvious abnormal health effects, Zhang says. All the same, “the most ideal case is, we want to shut off the enzyme.” And that may mean finding nonviral methods for getting Cas9 into cells, such as ferrying the protein with lipids or nanoparticles—delivery methods that biologists have long struggled to make work in living animals.

    Other long-standing obstacles to gene therapy will confront efforts using CRISPR, too. Depending on the disease, any gene-edited cells may eventually die and patients could have to be treated multiple times. Researchers using gene transfer and editing approaches are also both hindered by limits on how much DNA a viral vector can carry. Right now CRISPR researchers often must use two different viruses to get CRISPR’s components into cells, which is less efficient than a single vector.

    So what’s the bottom line?

    Gene therapists remain excited by CRISPR, in part because it could tackle many more inherited diseases than can be treated with gene transfer. Among them are certain immune diseases where the amount of the repaired protein must be precisely controlled. In other cases, such as sickle cell disease, patients won’t get completely well unless a defective protein is no longer made by their cells, so just adding a gene isn’t enough. “It opens up a lot of diseases to gene therapy because gene addition wasn’t going to work,” Dunbar says.

    After more than 2 decades of seeing their field through ups and downs, veterans of the gene therapy field are wary of raising expectations about CRISPR for treating diseases. “Whenever there’s a new technology, there’s a huge amount of excitement and everybody thinks it will be ready tomorrow to cure patients,” says gene therapy researcher Mark Kay of Stanford University in Palo Alto, California. “It’s going to take some time.”

    See the full article here .

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  • richardmitnick 2:12 pm on March 22, 2016 Permalink | Reply
    Tags: , , CRISPR,   

    From MIT: “Toward a better understanding of the brain” 

    MIT News
    MIT News

    March 22, 2016
    Anne Trafton | MIT News Office

    1
    “I was always interested in biology but I felt that it’s important to get a solid training in chemistry and physics,” Feng Zhang says. Photo: Bryce Vickmark

    Brain

    In 2011, about a month after joining the MIT faculty, Feng Zhang attended a talk by Harvard Medical School Professor Michael Gilmore, who studies the pathogenic bacterium Enteroccocus. The scientist mentioned that these bacteria protect themselves from viruses with DNA-cutting enzymes known as nucleases, which are part of a defense system known as CRISPR.

    “I had no idea what CRISPR was but I was interested in nucleases,” Zhang says. “I went to look up CRISPR, and that’s when I realized you might be able to engineer it for use for genome editing.”

    Zhang devoted himself to adapting the system to edit genes in mammalian cells and recruited new members to his nascent lab at the Broad Institute of MIT and Harvard to work with him on this project. In January 2013, they reported their success in the journal Science.

    Since then, scientists in fields from medicine to plant biology have begun using CRISPR to study gene function and investigate the possibility of correcting faulty genes that cause disease. Zhang now heads a lab of 19 scientists who continue to develop the system and pursue applications of genome editing, especially in neuroscience.

    “The goal is to try to make our lives better by developing new technologies and using them to understand biological systems so that we can improve our treatment of disease and our quality of life,” says Zhang, W. M. Keck Career Development Associate Professor in Biomedical Engineering and a member of MIT’s McGovern Institute for Brain Research. Zhang recently earned tenure in MIT’s Departments of Biological Engineering and Brain and Cognitive Sciences.

    Understanding the brain

    Growing up in Des Moines, Iowa, where his parents moved from China when he was 11, Zhang had plenty of opportunities to feed his interest in science. He participated in Science Bowl competitions and took special Saturday science classes, where he got his first introduction to molecular biology. Experiments such as extracting DNA from strawberries and transforming bacteria with genes for drug resistance whetted his appetite for genetic engineering, which was further stimulated by a showing of “Jurassic Park.”

    “That really caught my attention,” he recalls. “It didn’t seem that far-fetched. I guess that’s what makes it good science fiction. It kind of tantalizes your imagination.”

    As a sophomore in high school, Zhang began working with Dr. John Levy in a gene therapy lab at the Iowa Methodist Medical Center in Des Moines, where he studied green fluorescent protein (GFP). Scientists had recently figured out how to adapt this naturally occurring protein to tag and image proteins inside living cells. Zhang used it to track viral proteins within infected cells to determine how the proteins assemble to form new viruses. He also worked on a project to adapt GFP for a different purpose — protecting DNA from damage induced by ultraviolet light.

    At Harvard University, where he earned his undergraduate degree, Zhang majored in chemistry and physics and did research under the mentorship of Xiaowei Zhuang, a professor of chemistry and chemical biology. “I was always interested in biology but I felt that it’s important to get a solid training in chemistry and physics,” he says.

    While Zhang was at Harvard, a close friend was severely affected by a psychiatric disorder. That experience made Zhang think about whether such disorders could be approached just like cancer or heart disease, if only scientists knew more about their underlying causes.

    “The difference is we’re at a much earlier stage of understanding psychiatric diseases. That got me really interested in trying to understand more about how the brain works,” he says.

    At Stanford University, where Zhang earned his PhD in chemistry, he worked with Karl Deisseroth, who was just starting his lab with a focus on developing new technology for studying the brain. Zhang was the second student to join the lab, and he began working on a protein called channelrhodopsin, which he and Deisseroth believed held potential for engineering mammalian cells to respond to light.

    The resulting technique, known as optogenetics, has transformed biological research. Collaborating with Edward Boyden, a member of the Deisseroth lab who is now a professor at MIT, Zhang adapted channelrhodopsin so that it could be inserted into neurons and make them light-sensitive. Using this approach, neuroscientists can now selectively activate and de-activate specific neurons in the brain, allowing them to map brain circuits and investigate how disruption of those circuits causes disease.

    Better gene editing

    After leaving Stanford, Zhang spent a year as a junior fellow at the Harvard Society of Fellows, studying brain development with Professor Paola Arlotta and collaborating with Professor George Church. That’s when he began to focus on gene editing — a type of genetic engineering that allows researchers to selectively delete a gene or replace it with a new one.

    He began with zinc finger nucleases — enzymes that can be designed to target and cut specific DNA sequences. However, these proteins turned out to be challenging to work with, in part because it is so time-consuming to design a new protein for each possible DNA target.

    That led Zhang to experiment with a different type of nucleases known as transcription activator-like effector nucleases (TALENs), but these also proved laborious to work with. “Learning how to use them is a project on its own,” Zhang says.

    When he heard about CRISPR in early 2011, Zhang sensed that harnessing the natural bacterial process held the potential to solve many of the challenges associated with those earlier gene-editing techniques. CRISPR includes a nuclease called Cas9, which can be guided to the correct genetic target by RNA molecules known as guide strands. For each target, scientists need only design and synthesize a new RNA guide, which is much simpler than creating new TALEN and zinc finger proteins.

    Since his first CRISPR paper in 2013, Zhang’s lab has devised many enhancements to the original system, such as making the targeting more precise and preventing unintended cuts in the wrong locations. They also recently reported another type of CRISPR system based on a different nuclease called Cpf1, which is simpler and has unique features that further expand the genome editing toolbox.

    Zhang’s lab has become a hub for CRISPR research worldwide. It has shared CRISPR-Cas9 components in response to nearly 30,000 requests from academic laboratories around the world and has trained thousands of researchers in the use of CRISPR-Cas9 genome-editing technology through in-person events and online opportunities.

    His team is now working on creating animal models of autism, Alzheimer’s, and other neurological disorders, and in the long term, they hope to develop CRISPR for use in humans to potentially cure diseases caused by defective genes.

    “There are many genetic diseases that we don’t have any way of treating and this could be one way, but we still have to do a lot of work,” Zhang says.

    See the full article here .

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  • richardmitnick 5:31 pm on February 5, 2016 Permalink | Reply
    Tags: , CRISPR,   

    From SA: “The Embarrassing, Destructive Fight over Biotech’s Big Breakthrough” 

    Scientific American

    Scientific American

    February 4, 2016
    Stephen S. Hall

    The gene-editing technology known as CRISPR has spawned an increasingly unseemly brawl over who will reap the rewards.

    A defining moment in modern biology occurred on July 24, 1978, when biotechnology pioneer Robert Swanson, who had recently co-founded Genentech, brought two young scientists to dinner with Thomas Perkins, the legendary venture capitalist. As they stood outside Perkins’s magnificent mansion in Marin County, with its swimming pool and garden and a view of the Golden Gate Bridge, Swanson turned to his two young colleagues and said, “This is what we’re all working for.”

    That scene came to mind as I sorted through the tawdry verbal wreckage on social media and in print of the “debate” over CRISPR, the revolutionary new gene-editing technology. The current brouhaha, triggered by Eric Lander’s now-infamous essay in Cell called The Heroes of CRISPR, is the most entertaining food fight in science in years.

    The stakes are exceedingly high. CRISPR is the most important new technology to hit biology since recombinant DNA, which launched Genentech, made Swanson, along with his colleagues and investors, rich and brought molecular biology, long the province of academia, into the realm of celebrity and big money. In this context, the Cell essay has huge patent and prize implications. Lander has been accused of writing an incomplete and inaccurate history of the CRISPR story, burnishing the patent claims of the Broad Institute in Cambridge, Mass., (he is its director) and minimizing the contributions of rival scientists. A blogger has referred to him as “an evil genius at the height of his craft.” And George Church, a colleague at the Broad Institute, likens Lander to a figure out of a Greek tragedy. “The only person that could hurt him was himself,” he says. “He was invulnerable to anybody else’s sword.” And you thought scientists couldn’t talk smack.

    Spectators, scientific and otherwise, have followed this bitter dispute with fascination but the fight is destructive–and far from over. In waging a nasty public battle over CRISPR, the protagonists have given science a black eye for the reason Swanson suggested on that long-ago night in Marin County: money and glory. In waging a nasty public fight over CRISPR, they have already attracted the scrutiny of the mainstream press (The Washington Post and the Boston Globe Media’s STAT, to name two avid voyeurs), the scientific press and, oh boy, the Internet. The trash talking has undermined the public image of science, raised unflattering questions about the motives of scientists and institutions and, less obviously, fueled doubts about the judgment of leading scientific journals, which act as unofficial auditors of the billions of taxpayer dollars spent on biological research. The spat is like an escalating and increasingly ugly domestic dispute: no one wants outsiders to get involved but the screaming has gotten so loud that somebody has to call the cops. The fight over CRISPR is getting to that point. Woe be it to science if the politicians step in and use the fight as an excuse to rethink funding or the rules of technology transfer.

    The scientific story has deep roots. Scientists glimpsed the first hint of CRISPR biology in the 1980s and primitive forms of gene-editing arose in the 1990s. But a crucial leap occurred in 2012 when a group led by Jennifer Doudna of the University of California, Berkeley, and Emmanuelle Charpentier, now at the Max Planck Institute for Infection Biology in Berlin, demonstrated the possibility of simple CRISPR-based gene-editing to a broad audience of scientists with a paper in Science. The University of California and the University of Vienna filed for a patent, listing Doudna, Charpentier and other individuals. But the U.S. Patent and Trademark Office issued a patent in 2014 to Feng Zhang of the Broad Institute, which filed its application after Berkeley but requested expedited consideration. The University of California has challenged the validity of all the Broad patents (now numbering about a dozen) and the ensuing “interference” proceedings may allow another year of trash talking by scientists and bloggers alike. Meanwhile the protagonists—and their institutional proxies—continue to jockey for priority, prizes and reputation.

    Against this backdrop, Lander’s piece came as a shock. Lander is director of the Broad Institute and therefore someone with a very big dog in the patent fight. Maybe by calling it a “Perspective” the editors of Cell were signaling Lander’s obvious conflict of interest; nothing else in the article did.

    Let me note, first, that The Heroes of CRISPR is beautifully written. In addition to being a fabulous scientist and truly visionary thinker, Lander is a terrific communicator. His history reads at times like high-end magazine journalism (the story begins in Spain’s Costa Blanca, “where the beautiful coast and vast salt marshes have for centuries attracted vacationers, flamingoes and commercial salt producers”) with almost novelistic detail (Zhang, born in China and reared in Des Moines, has his eureka moment while “holed up” in a Miami hotel)—not your average journal prose. Lander’s account of the early work on CRISPR, often overlooked, is thorough, accurate and generous, according to people who know the history well. And it’s written as a feel-good story with an inspirational take-home message: People who work off the beaten track, in both the geographic and biological sense, often make dramatic contributions to the “remarkable ecosystem underlying scientific discovery,” he notes. Scientific breakthroughs are “ensemble acts” that unfold over many years. “It’s a wonderful lesson for the general public,” Lander concludes, “as well as for a young person contemplating a life in science.”

    Beautifully put. So why did the Twitterverse go radioactive on Lander within hours of the article’s publication on January 14?

    As I often suggest to students in my science journalism classes, just because a story is beautifully written doesn’t mean that it is true—in whole or in part. Judging from the firestorm of criticism, “The Heroes of CRISPR” falls short on a number of issues, beginning with this awkward money-tinged paradox: If the CRISPR story (and science in general) is such a beautiful ensemble activity, why is there only one name on the Broad Institute’s patent? Well, patents have to do with money, and money turns a lot of beautiful scientific stories into ugly legal narratives.

    Case in point: in 1979, a year after Bob Swanson’s pep talk to his Genentech biologists, a biologist then at Columbia University named Michael Wigler published a very clever method (called “co-transformation”) for smuggling genes into eukaryotic cells; the university filed a patent application in 1980, with Wigler and two colleagues as inventors, and received the first of several patents in 1983. Like CRISPR, the technique may sound esoteric but biologists (and companies) quickly recognized its value, and Columbia ultimately reaped nearly $800 million from those patents. (Other, unofficial estimates run between $1 billion and $1.5 billion.) Columbia became so enamored of the revenue stream that it resorted to several controversial tactics, including having a U.S. senator try to extend the patent by slipping language into an agricultural bill. These maneuvers prompted an uproar and were later characterized, by historians of genomics Robert Cook-Deegan and Alessandra Colaianni, both then at Duke University, as “behavior unbecoming a nonprofit academic institution.”

    Wigler, who now runs a lab at Cold Spring Harbor Laboratory, says of the Columbia patent: “Of course it’s had an impact on institutions, because institutions are desperate for money.” That’s why the University of California and the Broad Institute (a joint venture of Harvard University and Massachusetts Institute of Technology) will fight fiercely—“red in tooth and claw,” you might say—to claim intellectual property on CRISPR.

    Many readers (including me) interpreted Lander’s elegant history of CRISPR as a calculated attempt to elevate the intellectual contribution of Zhang (the Broad Institute scientist who is recognized, for the moment, by the patent office as the lone “inventor” of CRISPR) as it minimizes the contributions of Doudna and Charpentier. (Zhang’s discovery narrative is long, detailed and colorful; Doudna’s appearance comes in the middle of a paragraph, and her work doesn’t get nearly the same star treatment.) In other words, this beautifully crafted history can also be read as a patent brief in disguise. (A blog by science historian Nathaniel Comfort shrewdly deconstructs the rhetoric used by Lander to advance Broad’s interests. Inexplicably, Cell didn’t even mention Lander’s flagrant conflict of interest (an instance of editorial neglect to which we’ll return later).

    Both Doudna and Charpentier quickly posted frostily brief comments to PubMed Commons; Doudna claimed the description of her lab’s work was “factually incorrect,” and Charpentier characterized her part of the story as “incomplete and inaccurate.” Church, whose Harvard lab published on the utility of CRISPR gene-editing in mammalian cells at the same time as Zhang’s, disputed Lander’s history as well in press accounts. When I spoke with Church about a week after the Cell article came out, he was not shy about itemizing. “Normally I’m not so nitpicky about all these errors,” he said. “But as soon as I saw that they [Lander and Cell] were not giving the young people, the people who actually did the work, and Jennifer and Emmanuelle, adequate credit, I just said, ‘No, I have to correct what I know to be false.’” (Lander was “delighted” to append Church’s clarifications to the Cell article). Church acknowledged that the essay was “exquisitely crafted,” but crafted, in his view, with an ulterior motive. “It was like, ‘I’m going to prove my point of view,’” he said. But according to Church, Lander may have achieved the exact opposite effect. “I think Jennifer and Emmanuelle deserve a lot of credit,” he said. “And the more you try to take it from them, the more people want to give it to them.”

    In truth, there are a lot of moving parts and proxies in this messy battle. The hostilities involve institutions (M.I.T. and Harvard versus University of California), gender (Doudna, Charpentier, Zhang), geography (east versus west coast) and what you might call über-institutions (the Broad Institute, which has become an empire of genomic research under Lander’s direction, especially after his leading role in the Human Genome Project, versus the Howard Hughes Medical Institute, whose president, Robert Tjian, is based at Berkeley and has co-authored at least one CRISPR paper with Doudna, also an HHMI investigator). Probably because of this combustible mix of interests, the debate over the Cell article has become especially nasty; whatever used to be the line of decorum in scientific debate, it was breached within 24 hours after the Cell article appeared.

    Some viewed Lander’s history as a gender diss. The title of a post on the Web site Jezebel says it all: How One Man Tried to Write Women Out of CRISPR, the Biggest Biotech Innovation in Decades. Others saw it as shameless politicking for a Nobel Prize.

    And a lot of the invective has been surprisingly personal. Michael Eisen, an HHMI researcher at Berkeley, has been particularly outspoken in his blog. The Cell essay was “an elaborate lie,” Eisen wrote on January 25, and his attack didn’t stop there.

    Lander is in Antarctica and unavailable for comment, according to a Broad Institute spokesperson. But in an e-mail to the Broad staff on January 28 he reiterated his pride in writing the essay and added: “Needless to say, ‘Perspective’ articles are personal opinions. Not everyone will fully agree with anyone else’s point of view. In the end, we come to understand science only by integrating a diverse range of thoughtfully expressed perspectives. And, when scientific discovery is also the subject of patent disputes (as is the case with U.C. Berkeley and Broad–M.I.T.), intellectual disagreements can, as here, give rise to vigorous online discussion.” As for the conflict-of-interest issue, Broad spokesperson Lee McGuire noted that Lander had previously “disclosed the fact that he has no personal financial interest and that the institute he represents does license CRISPR technologies.”

    The dirty truth is that long before Lander’s Cell article the scientific community has been watching this food fight—for patent dollars, for credit, for prizes—with increasing dismay. Both Zhang and Doudna have been subtly lobbying for recognition in what one scientist characterized to me, dismissively, as “their little Nobel talks—they don’t give seminars anymore.” Doudna, Charpentier and Zhang are all outstanding researchers and very likable people but they appear caught up in the vortex of scientific politics and recognition spin. If it were your work and someone was trying to devalue it, you’d defend it to the hilt, too. But the ongoing drama is not a good look for science, and some of the “heroes of CRISPR” are wearing out their welcome on the public stage. “This is not David versus Goliath,” one disgusted scientist told me recently. “This is Goliath against Goliath. These two camps deserve each other, and they can bully each other into oblivion.”

    Why would such a shrewd and strategic thinker like Lander tempt such a public backlash by writing such a cleverly slanted history? Perhaps his ultimate audience was not Cell’s readers nor even the scientific community at large but rather a very small (and select) group of readers in Alexandria, Va. A gifted writer, Lander set out to produce a seemingly neutral and magnanimous history of CRISPR that even an examiner at the U.S. Patent and Trade Office could understand. (If this sounds condescending, consider how Wigler summarizes 35-plus years of dealing with the patent system: “My general experience with the patent office is that they don’t get it. They don’t understand this stuff.”)

    The Lander article has inflicted some surprising collateral damage, notably to scientific publishing itself. Cell’s decision to publish the article, despite the Broad’s clear financial interest in the patent dispute, invited withering criticism. (The journal stated that it “regularly” evaluates its policies, and “will include” in that process the role of institutional conflicts of interest.) And if CRISPR is “the century’s biggest biotech innovation,” as a blogger for The Washington Post recently noted, what does it say about the quality of scientific journals that in at least 10 instances “seminal papers,” according to Lander’s Cell article, were rejected by journals like Nature, Proceedings of the National Academy of Sciences and even Cell itself? In many cases, editors at these journals did not even send out the articles for peer review. Virtually all this research is paid for at least in part by public money, which raises an inconvenient question: Is the public interest served by journals that don’t even recognize scientific excellence? Does Cell even give a hoot about public perception? Here’s what one prominent scientist told me about Cell’s handling of the entire episode: “All they care about is how many times the article is cited in their citation index.” That and the traffic on Twitter.

    That may hint at why the CRISPR dispute is so different, and so dangerous to the scientific community. Trash talking has been a part of science for centuries; Newton’s seemingly magnanimous remark that he stood “on the shoulders of giants” was, to the contrary, likely understood by his contemporaries to be a disparaging reference to the short stature of his main rival, Robert Hooke. But invective today gets amplified and disseminated so rapidly that it assumes a public life of its own, and scientific spats become a reality show complete with egos, self-promotion, greed and Machiavellian stratagems dissected in blogs, on social media and on bulletin boards.

    And then there’s the influence of money. Since the summer of 1978 biotechnology has bestowed untold riches on companies, institutions and individual biologists. It has produced thrilling science and some wonderful (albeit pricey) new medicines. But it has also slowly eroded boundaries between appropriate and inappropriate behavior. Scientific narratives used to be cast in the past tense, about what had been accomplished; now the storytelling is in the future tense to raise venture capital (or, in the case of ‘Heroes,” in what might be called the past imperfect to advance a patent claim). Hype used to be frowned on; now it is part of every business plan. Since at least the 1990s biotech companies have tried to influence university research, and it is a commonplace that the pharmaceutical industry dictates the terms of much academic clinical research. Students, already demoralized by scarce funding and no jobs, fret over whether basic research (of the sort that produced CRISPR in the first place) will be as esteemed as “patentable” work—and if their names will even be included on the patent. And the red flags that used to signal conflicts of interest are so frayed that you can essentially see right through them. It’s not that Cell should have had a stricter policy about conflicts of interest, it’s that a protagonist in the patent dispute probably shouldn’t have attempted to write a history of CRISPR in the first place. After the Lander article was published Cell posted a statement on its Web site saying Lander had indeed communicated that his institutional affiliations—Broad, M.I.T. and Harvard—had patents and patent applications related to CRISPR but that the journal only considers “personal” conflicts of interest.

    There is currently a lull in the CRISPR hostilities; no one has flamed anyone, by my count, in the last 72 hours. But that probably won’t last long. The patent interference, in which University of California lawyers will probably claim that its scientists invented CRISPR gene-editing and also applied for a patent before Broad, will be hotly contested. Maybe this little pause is an opportunity for a reset—a chance for the scientific community to acknowledge that the CRISPR system, as some have quietly suggested all along, was actually “invented” by bacteria eons ago as an ingenious immune response to viral infection, and that its rediscovery was accomplished by so many heroic (if you will) hands and with so much public coin that the technology ultimately belongs in the public commons and should not be patented and…

    …Sorry, I got a little carried away there. Yes, it would be nice if the transformative power of CRISPR remained in the public domain; maybe we could even invent a new prize—the Rashomon Prize!—that recognizes all the key players, no matter how contradictory or self-serving their stories. But in the current ecosystem of biology, where institutions are indeed desperate for money and the rules of the game create winner-take-all slugfests, that is very unlikely to happen.

    See the full article here .

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  • richardmitnick 11:51 am on December 22, 2015 Permalink | Reply
    Tags: , CRISPR, ,   

    From New Scientist: “CRISPR will make 2016 the year of gene-edited organisms” 

    NewScientist

    New Scientist

    15 December 2015
    Next year preview
    Michael Le Page

    1
    Image credit: Dr Yorgos Nikas/Science Photo Library

    Will the first gene-edited baby be born in 2016? Let’s hope not. It is far from clear it can be done safely – although technically it is now possible. Gene editing with the new method known as CRISPR is so cheap, easy and effective that a few scientists with the appropriate expertise could tweak one or more genes in a human embryo before it is implanted in a woman’s womb.

    What 2016 will undoubtedly bring is a lot more gene-edited organisms. CRISPR works well in everything from butterflies to monkeys. It has already been used to create extra-muscular beagle dogs and sheep; long-haired goats; and pigs immune to common diseases. Next up could be hypoallergenic pet dogs and cats, cattle resistant to TB, or chickens that don’t get bird flu.

    But whether any of these make it out of the lab in the next 12 months depends on the regulators. Gene editing can add new pieces of DNA, as in conventional genetic engineering, so any living thing altered in this way is bound in many countries by strict regulations on genetically modified organisms. Getting approval to sell modified animals takes a lot of time and money.

    But gene editing can also be used to make changes to existing genes – tweaks that are indistinguishable from naturally occurring gene variants. The mutation that made the beagles more muscly already exists in dog breeds like the bully whippet.

    In theory, this kind of gene editing should be exempt from regulation. If the regulators agree, this could be the year that people start eating, drinking or wearing products from gene-edited farm animals and plants, or buy the first gene-edited pets.

    See the full article here .

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  • richardmitnick 7:45 am on August 10, 2015 Permalink | Reply
    Tags: , , , CRISPR,   

    From Cosmos: “Modifying human immune cells to fight cancer” 

    Cosmos Magazine bloc

    COSMOS

    10 Aug 2015
    Viviane Richter and Elizabeth Finkel

    1
    Coloured scanning electron micrograph of T cells (pink) attacking a cancer cell. Editing T cells’ genes could soon enhance their cancer-attacking abilities.Credit: Science Photo Library / Getty Images

    Immune cells known as T cells are formidable fighters against cancer and HIV.

    2
    Scanning electron micrograph of a human T cell

    But they can be outsmarted by these foes. Now researchers at the University of California, San Francisco have figured out how to help T cells fight back using the latest gene editing technique called CRISPR. The method was published in Proceedings of the National Academy of Sciences in July. “This technique opens a lot of doors for the field,” says lead author Alexander Marson.

    Scientists have been tinkering with genes since the early 1970s to create faster growing pigs or herbicide-resistant crops. But the techniques had poor precision. Tens of thousands of individuals had to be tinkered with to achieve the required edits and usually those edits would be inserted on the wrong pages of the DNA text.

    To reliably manipulate specific genes to fight human disease, pinpoint precision is required.

    2
    The CRISPR-CAS9 gene editing machinery from the Streptococcus pyogenes bacterium. RNA strands (blue) guide CRISPR to a targeted stretch of DNA, where it can snip out a specific gene. Credit: MOLEKUUL / SCIENCE PHOTO LIBRARY / Getty Images

    Which is exactly what the CRISPR gene editing technique offers. It’s no surprise that since scientists first discovered CRISPR in bacteria three years ago, it has taken the world by storm. Microbes evolved CRISPR to edit viruses out of their DNA. Now it’s been used to precisely edit everything from the DNA of crops to editing the HIV virus out of human DNA. Last April, Chinese researchers used it to edit the DNA of a human embryo – a move that created a storm of controversy. Till now tampering with the DNA of an embryo was considered out of bounds.

    But tampering with T cells is not likely to attract bad press. In particular, blood borne T cells form the major defence against viruses and cancer. Or when they misbehave, they cause auto-immune diseases such as Type 1 Diabetes. Controlling these cells by rewriting their DNA could mean a cure for incurable diseases. It’s all very doable: simply filter T cells from a person’s blood, edit their DNA and return them to the individual to do their job.

    The challenge is delivering the CRISPR machinery into T cells. Usually it’s done by packaging CRISPR into a harmless virus that ferries it into cells. CRISPR itself then acts like a guided missile, homing in on a precise stretch of the DNA code. (The guide that targets the DNA is a small piece of RNA. The missile is a shredding protein called Cas 9.) But so far, getting this guided missile inside the cells has been at the very low end of precision and efficiency. If only a tiny percentage of T cells can be engineered to resist HIV or fight cancer, it might hardly be worth the effort.

    So Marson’s team tried brute force. They zapped T cells from healthy donors with an electric current. This made temporary holes in the cells’ membrane, big enough for the intact CRISPR machinery to pop through. They managed to edit the DNA in 20 percent of T cells, a “huge” leap in efficiency according to Marco Herold, molecular biologist at Melbourne’s Walter and Eliza Hall Institute of Medical Research.

    Yet in contrast to their brute force entry, they were able to achieve very fine editing, changing individual letters of the cell’s DNA for the first time, as opposed to inserting or deleting large chunks. For instance they altered a doorway used by HIV known as CXCR4, so the deadly virus would not be able to enter and infect these T cells.

    They were also able to edit a gene called PD-1. Its role is to tell T cells to lay down their weapons so they don’t for instance attack normal cells of the body. But crafty cancer cells have learned how to give this same command to PD-1, so the T-cells lay down their arms in the vicinity of cancer cells. By editing the PD-1 gene of the T cells, the researchers should be able to turn T cells back into a fighting force against cancer. Simular approaches are successfully being trialled in cancer therapy employing antibodies to turn off the PD-1 signal.

    However the editing approach raises some concerns since T cells are long-term residents – sometimes remaining in the body for years. And if they are unleashed against cancer, one has to be sure they will not then go on to attack normal cells. “The danger is to create something you can’t control,” says Herold.

    “We have to work ahead to figure out how to ensure safety” of T cells, Marson agrees. “But it’s an exciting time for cell-based therapies.

    See the full article here.

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