Researcher: Feng Zhang

Rapid test for Covid-19 shows improved sensitivity

by Anne Trafton |

Since the start of the Covid-19 pandemic, researchers at MIT and the Broad Institute of MIT and Harvard, along with their collaborators at the University of Washington, Fred Hutchinson Cancer Research Center, Brigham and Women’s Hospital, and the Ragon Institute, have been working on a CRISPR-based diagnostic for Covid-19 that can produce results in 30 minutes to an hour, with similar accuracy as the standard PCR diagnostics now used.

The new test, known as STOPCovid, is still in the research stage but, in principle, could be made cheaply enough that people could test themselves every day. In a study appearing today in the New England Journal of Medicine, the researchers showed that on a set of patient samples, their test detected 93 percent of the positive cases as determined by PCR tests for Covid-19.

“We need rapid testing to become part of the fabric of this situation so that people can test themselves every day, which will slow down outbreak,” says Omar Abudayyeh, an MIT McGovern Fellow working on the diagnostic.

Abudayyah is one of the senior authors of the study, along with Jonathan Gootenberg, a McGovern Fellow, and Feng Zhang, a core member of the Broad Institute, investigator at the MIT McGovern Institute and Howard Hughes Medical Institute, and the James and Patricia Poitras ’63 Professor of Neuroscience at MIT. The first authors of the paper are MIT biological engineering graduate students Julia Joung and Alim Ladha in the Zhang lab.

A streamlined test

Zhang’s laboratory began collaborating with the Abudayyeh and Gootenberg laboratory to work on the Covid-19 diagnostic soon after the SARS-CoV-2 outbreak began. They focused on making an assay, called STOPCovid, that was simple to carry out and did not require any specialized laboratory equipment. Such a test, they hoped, would be amenable to future use in point-of-care settings, such as doctors’ offices, pharmacies, nursing homes, and schools.

“We developed STOPCovid so that everything could be done in a single step,” Joung says. “A single step means the test can be potentially performed by nonexperts outside of laboratory settings.”

In the new version of STOPCovid reported today, the researchers incorporated a process to concentrate the viral genetic material in a patient sample by adding magnetic beads that attract RNA, eliminating the need for expensive purification kits that are time-intensive and can be in short supply due to high demand. This concentration step boosted the test’s sensitivity so that it now approaches that of PCR.

“Once we got the viral genomes onto the beads, we found that that could get us to very high levels of sensitivity,” Gootenberg says.

Working with collaborators Keith Jerome at Fred Hutchinson Cancer Research Center and Alex Greninger at the University of Washington, the researchers tested STOPCovid on 402 patient samples — 202 positive and 200 negative — and found that the new test detected 93 percent of the positive cases as determined by the standard CDC PCR test.

“Seeing STOPCovid working on actual patient samples was really gratifying,” Ladha says.

They also showed, working with Ann Woolley and Deb Hung at Brigham and Women’s Hospital, that the STOPCovid test works on samples taken using the less invasive anterior nares swab. They are now testing it with saliva samples, which could make at-home tests even easier to perform. The researchers are continuing to develop the test with the hope of delivering it to end users to help fight the COVID-19 pandemic.

“The goal is to make this test easy to use and sensitive, so that we can tell whether or not someone is carrying the virus as early as possible,” Zhang says.

The research was funded by the National Institutes of Health, the Swiss National Science Foundation, the Patrick J. McGovern Foundation, the McGovern Institute for Brain Research, the Massachusetts Consortium on Pathogen Readiness Evergrande Covid-19 Response Fund, the Mathers Foundation, the Howard Hughes Medical Institute, the Open Philanthropy Project, J. and P. Poitras, and R. Metcalfe.

 

FULL PAPER AT NEJM

New molecular therapeutics center established at MIT’s McGovern Institute

by Julie Pryor |

More than one million Americans are diagnosed with a chronic brain disorder each year, yet effective treatments for most complex brain disorders are inadequate or even nonexistent.

A major new research effort at MIT’s McGovern Institute aims to change how we treat brain disorders by developing innovative molecular tools that precisely target dysfunctional genetic, molecular, and circuit pathways.

The K. Lisa Yang and Hock E. Tan Center for Molecular Therapeutics in Neuroscience was established at MIT through a $28 million gift from philanthropist Lisa Yang and MIT alumnus Hock Tan ’75. Yang is a former investment banker who has devoted much of her time to advocacy for individuals with disabilities and autism spectrum disorders. Tan is President and CEO of Broadcom, a global technology infrastructure company. This latest gift brings Yang and Tan’s total philanthropy to MIT to more than $72 million.

Lisa Yang (center) and MIT alumnus Hock Tan ’75 with their daughter Eva (far left) pictured at the opening of the Hock E. Tan and K. Lisa Yang Center for Autism Research in 2017. Photo: Justin Knight

“In the best MIT spirit, Lisa and Hock have always focused their generosity on insights that lead to real impact,” says MIT President L. Rafael Reif. “Scientifically, we stand at a moment when the tools and insights to make progress against major brain disorders are finally within reach. By accelerating the development of promising treatments, the new center opens the door to a hopeful new future for all those who suffer from these disorders and those who love them. I am deeply grateful to Lisa and Hock for making MIT the home of this pivotal research.”

Engineering with precision

Research at the K. Lisa Yang and Hock E. Tan Center for Molecular Therapeutics in Neuroscience will initially focus on three major lines of investigation: genetic engineering using CRISPR tools, delivery of genetic and molecular cargo across the blood-brain barrier, and the translation of basic research into the clinical setting. The center will serve as a hub for researchers with backgrounds ranging from biological engineering and genetics to computer science and medicine.

“Developing the next generation of molecular therapeutics demands collaboration among researchers with diverse backgrounds,” says Robert Desimone, McGovern Institute Director and Doris and Don Berkey Professor of Neuroscience at MIT. “I am confident that the multidisciplinary expertise convened by this center will revolutionize how we improve our health and fight disease in the coming decade. Although our initial focus will be on the brain and its relationship to the body, many of the new therapies could have other health applications.”

There are an estimated 19,000 to 22,000 genes in the human genome and a third of those genes are active in the brain–the highest proportion of genes expressed in any part of the body.

Variations in genetic code have been linked to many complex brain disorders, including depression and Parkinson’s. Emerging genetic technologies, such as the CRISPR gene editing platform pioneered by McGovern Investigator Feng Zhang, hold great potential in both targeting and fixing these errant genes. But the safe and effective delivery of this genetic cargo to the brain remains a challenge.

Researchers within the new Yang-Tan Center will improve and fine-tune CRISPR gene therapies and develop innovative ways of delivering gene therapy cargo into the brain and other organs. In addition, the center will leverage newly developed single cell analysis technologies that are revealing cellular targets for modulating brain functions with unprecedented precision, opening the door for noninvasive neuromodulation as well as the development of medicines. The center will also focus on developing novel engineering approaches to delivering small molecules and proteins from the bloodstream into the brain. Desimone will direct the center and some of the initial research initiatives will be led by Associate Professor of Materials Science and Engineering Polina Anikeeva; Ed Boyden, the Y. Eva Tan Professor in Neurotechnology at MIT; Guoping Feng, the James W. (1963) and Patricia T. Poitras Professor of Brain and Cognitive Sciences at MIT; and Feng Zhang, James and Patricia Poitras Professor of Neuroscience at MIT.

Building a research hub

“My goal in creating this center is to cement the Cambridge and Boston region as the global epicenter of next-generation therapeutics research. The novel ideas I have seen undertaken at MIT’s McGovern Institute and Broad Institute of MIT and Harvard leave no doubt in my mind that major therapeutic breakthroughs for mental illness, neurodegenerative disease, autism and epilepsy are just around the corner,” says Yang.

Center funding will also be earmarked to create the Y. Eva Tan Fellows program, named for Tan and Yang’s daughter Eva, which will support fellowships for young neuroscientists and engineers eager to design revolutionary treatments for human diseases.

“We want to build a strong pipeline for tomorrow’s scientists and neuroengineers,” explains Hock Tan. “We depend on the next generation of bright young minds to help improve the lives of people suffering from chronic illnesses, and I can think of no better place to provide the very best education and training than MIT.”

The molecular therapeutics center is the second research center established by Yang and Tan at MIT. In 2017, they launched the Hock E. Tan and K. Lisa Yang Center for Autism Research, and, two years later, they created a sister center at Harvard Medical School, with the unique strengths of each institution converging toward a shared goal: understanding the basic biology of autism and how genetic and environmental influences converge to give rise to the condition, then translating those insights into novel treatment approaches.

All tools developed at the molecular therapeutics center will be shared globally with academic and clinical researchers with the goal of bringing one or more novel molecular tools to human clinical trials by 2025.

“We are hopeful that our centers, located in the heart of the Cambridge-Boston biotech ecosystem, will spur further innovation and fuel critical new insights to our understanding of health and disease,” says Yang.

 

SHERLOCK-based one-step test provides rapid and sensitive COVID-19 detection 

by Sabbi Lall |

A team of researchers at the McGovern Institute for Brain Research at MIT, the Broad Institute of MIT and Harvard, the Ragon Institute, and the Howard Hughes Medical Institute (HHMI) has developed a new diagnostics platform called STOP (SHERLOCK Testing in One Pot) COVID. The test can be run in an hour as a single-step reaction with minimal handling, advancing the CRISPR-based SHERLOCK diagnostic technology closer to a point-of-care or at-home testing tool. The test has not been reviewed or approved by the FDA and is currently for research purposes only.

The team began developing tests for COVID-19 in January after learning about the emergence of a new virus which has challenged the healthcare system in China. The first version of the team’s SHERLOCK-based COVID-19 diagnostics system is already being used in hospitals in Thailand to help screen patients for COVID-19 infection.

The ability to test for COVID-19 at home, or even in pharmacies or places of employment, could be a game-changer for getting people safely back to work and into their communities.

The new test is named “STOPCovid” and is based on the STOP platform. In research it has been shown to enable rapid, accurate, and highly sensitive detection of the COVID-19 virus SARS-CoV-2 with a simple protocol that requires minimal training and uses simple, readily-available equipment, such as test tubes and water baths. STOPCovid has been validated in research settings using nasopharyngeal swabs from patients diagnosed with COVID-19. It has also been tested successfully in saliva samples to which SARS-CoV-2 RNA has been added as a proof-of-principle.

The team is posting the open protocol today on a new website, STOPCovid.science. It is being made openly available in line with the COVID-19 Technology Access Framework organized by Harvard, MIT, and Stanford. The Framework sets a model by which critically important technologies that may help prevent, diagnose, or treat COVID-19 infections may be deployed for the greatest public benefit without delay.

There is an urgent need for widespread, accurate COVID-19 testing to rapidly detect new cases, ideally without the need for specialized lab equipment. Such testing would enable early detection of new infections and drive effective “test-trace-isolate” measures to quickly contain new outbreaks. However, current testing capacity is limited by a combination of requirements for complex procedures and laboratory instrumentation and dependence on limited supplies. STOPCovid can be performed without RNA extraction, and while all patient tests have been performed with samples from nasopharyngeal swabs, preliminary experiments suggest that eventually swabs may not be necessary. Removing these barriers could help enable broad distribution.

“The ability to test for COVID-19 at home, or even in pharmacies or places of employment, could be a game-changer for getting people safely back to work and into their communities,” says Feng Zhang, a co-inventor of the CRISPR genome editing technology, an investigator at the McGovern Institute and HHMI, and a core member at the Broad Institute. “Creating a point-of-care tool is a critically important goal to allow timely decisions for protecting patients and those around them.”

To meet this need, Zhang, McGovern Fellows Omar Abudayyeh and Jonathan Gootenberg, and colleagues initiated a push to develop STOPCovid. They are sharing their findings and packaging reagents so other research teams can rapidly follow up with additional testing or development. The group is also sharing data on the StopCOVID.science website and via a submitted preprint. The website is also a hub where the public can find the latest information on the team’s developments.

McGovern Institute Fellows Jonathan Gootenberg (far left) Omar Abudayyeh and have developed a CRISPR research tool to detect COVID-19 with McGovern Investigator Feng Zhang (far right).
Credit: Justin Knight

How it works

The STOPCovid test combines CRISPR enzymes, programmed to recognize signatures of the SARS-CoV-2 virus, with complementary amplification reagents. This combination allows detection of as few as 100 copies of SARS-CoV-2 virus in a sample. As a result, the STOPCovid test allows for rapid, accurate, and highly sensitive detection of COVID-19 that can be conducted outside clinical laboratory settings.

STOPCovid has been tested on patient nasopharyngeal swab in parallel with clinically-validated tests. In these head-to-head comparisons, STOPCovid detected infection with 97% sensitivity and 100% specificity. Results appear on an easy-to-read strip that is akin to a pregnancy test, in the absence of any expensive or specialized lab equipment. Moreover, the researchers spiked mock SARS-CoV-2 genomes into healthy saliva samples and showed that STOPCovid is capable of sensitive detection from saliva, which would obviate the need for swabs in short supply and potentially make sampling much easier.

“The test aims to ultimately be simple enough that anyone can operate it in low-resource settings, including in clinics, pharmacies, or workplaces, and it could potentially even be put into a turn-key format for use at home,” says Abudayyeh.

Gootenberg adds, “Since STOPCovid can work in less than an hour and does not require any specialized equipment, and if our preliminary results from testing synthetic virus in saliva bear out in patient samples, it could address the need for scalable testing to reopen our society.”

The STOPCovid team during a recent zoom meeting. Image: Omar Abudayyeh

Importantly, the full test — both the viral genome amplification and subsequent detection — can be completed in a single reaction, as outlined on the website, from swabs or saliva. To engineer this, the team tested a number of CRISPR enzymes to find one that works well at the same temperature needed by the enzymes that perform the amplification. Zhang, Abudayyeh, Gootenberg and their teams, including graduate students Julia Joung and Alim Ladha, settled on a protein called AapCas12b, a CRISPR protein from the bacterium Alicyclobacillus acidophilus, responsible for the “off” taste associated with spoiled orange juice. With AapCas12b, the team was able to develop a test that can be performed at a constant temperature and does not require opening tubes midway through the process, a step that often leads to contamination and unreliable test results.

Information sharing and next steps

The team has prepared reagents for 10,000 tests to share with scientists and clinical collaborators for free around the world who want to evaluate the STOPCovid test for potential diagnostic use, and they have set up a website to share the latest data and updates with the scientific and clinical community. Kits and reagents can also be requested via a form on the website.

Acknowledgments: Patient samples were provided by Keith Jerome, Alex Greninger, Robert Bruneau, Mee-li W. Huang, Nam G. Kim, Xu Yu, Jonathan Li, and Bruce Walker. This work was supported by the Patrick J. McGovern Foundation and the McGovern Institute for Brain Research. F.Z is also supported by the NIH (1R01- MH110049 and 1DP1-HL141201 grants); Mathers Foundation; the Howard Hughes Medical Institute; Open Philanthropy Project; J. and P. Poitras; and R. Metcalfe.

Declaration of conflicts of interest: F.Z., O.O.A., J.S.G., J.J., and A.L. are inventors on patent applications related to this technology filed by the Broad Institute, with the specific aim of ensuring this technology can be made freely, widely, and rapidly available for research and deployment. O.O.A., J.S.G., and F.Z. are co-founders, scientific advisors, and hold equity interests in Sherlock Biosciences, Inc. F.Z. is also a co-founder of Editas Medicine, Beam Therapeutics, Pairwise Plants, and Arbor Biotechnologies.

How We Feel app to track spread of COVID-19 symptoms

by Sabbi Lall |

A major challenge with containing the spread of COVID-19 in many countries, has been an ability to quickly detect infection. Feng Zhang, along with Pinterest CEO Ben Silberman, and collaborators across scientific and medical disciplines, are coming together to launch an app called How We Feel, that will allow citizen scientists to self-report symptoms.

“It is so important to find a way to connect scientists to fight this pandemic,” explained Zhang. We wanted to find a fast and agile way to ultimately build a dynamic picture of symptoms associated with the virus.”

Designed to help scientists track and stop the spread of the novel coronavirus by creating an exchange of information between the citizens and scientists at scale, the new How We Feel app does just this. The app lets people self-report symptoms in 30 seconds or less and see how others in their area are feeling. To protect user privacy, the app explicitly does not require an account sign in, and doesn’t ask for identifying information such as the user’s name, phone number, or email address before they donate their data. Reporting symptoms only takes about 30 seconds, but the data shared by users has the potential to reveal and even predict outbreak hotspots, potentially providing insight into the spread and progression of COVID-19. To further contribute to the fight against COVID-19, Ben and Divya Silbermann will donate a meal to Feeding America for every download of the How We Feel app—up to 10 million meals.

The app was created by the How We Feel Project, a nonprofit collaboration between Silbermann, doctors, and an interdisciplinary group of researchers including Feng Zhang, investigator at the McGovern Institute for Brain Research, Broad Institute, and the James and Patricia Poitras Professor of Neuroscience at MIT. Other institutions currently involved include Harvard University T.H. Chan School of Public Health and Faculty of Arts and Sciences, University of Pennsylvania, Stanford University, University of Maryland School of Medicine, and the Weizmann Institute of Science.

Silbermann partnered closely with Feng Zhang, best known for his work on CRISPR, a pioneering gene-editing technique designed to treat diseases. Zhang and Silbermann first met in high school in Iowa. As the outbreak grew in the US, they called each other to figure out how the fields of biochemistry and technology could come together to find a solution for the lack of reliable health data from testing.

“Since high school, my friend Feng Zhang and I have been talking about the potential of the internet to connect regular people and scientists for the public good,” said Ben Silbermann, co-founder and CEO of, Pinterest. “When we saw how quickly COVID-19 was spreading, it felt like a critical moment to finally build that bridge between citizens and scientists that we’ve always wanted. I believe we’ve done that with How We Feel.”

Silbermann and Zhang formed the new HWF nonprofit because they believed a fully independent organization with a keen understanding of the needs of doctors and researchers should develop and manage the app. Now, they’re looking for opportunities to collaborate globally. Zhang is working to organize an international consortium of researchers from 11 countries that have developed similar health status surveys. The consortium is called the Coronavirus Census Collective (CCC).

The How We Feel app is available for download today in the US on iOS and Android, and via the web at http://www.howwefeel.org.

Enabling coronavirus detection using CRISPR-Cas13: An open-access SHERLOCK research protocol

by McGovern Institute |

The recent coronavirus (COVID-19) outbreak presents enormous challenges for global health. To aid the global effort, Broad Institute of MIT and Harvard, the McGovern Institute for Brain Research at MIT, and our partner institutions have committed to freely providing information that may be helpful, including by sharing information that may be able to support the development of potential diagnostics.

As part of this effort, Feng Zhang, Omar Abudayyeh, and Jonathan Gootenberg have developed a research protocol, applicable to purified RNA, that may inform the development of CRISPR-based diagnostics for COVID-19.

This initial research protocol is not a diagnostic test and has not been tested on patient samples. Any diagnostic would need to be developed and validated for clinical use and would need to follow all local regulations and best practices.

The research protocol provides the basic framework for establishing a SHERLOCK-based COVID-19 test using paper strips.

The team welcomes researchers to contact them for assistance or guidance and can provide a starter kit to test this system, as available, for researchers working with COVID-19 samples.

The SHERLOCK protocol

The CRISPR-Cas13-based SHERLOCK system has been previously shown to accurately detect the presence of a number of different viruses in patient samples. The system searches for unique nucleic acid signatures and uses a test strip similar to a pregnancy test to provide a visual readout. After dipping a paper strip into a prepared sample, a line appears on the paper to indicate whether the virus is present.

Using synthetic COVID-19 RNA fragments, the team designed and tested two RNA guides that recognize two signatures of COVID-19. When combined with the Cas13 protein, these form a SHERLOCK system capable of detecting the presence of COVID-19 viral RNA.

The research protocol involves three steps. It can be used with the same RNA samples that have been extracted for current qPCR tests:

  1. Incubate extracted RNA with isothermal amplification reaction for 25 min at 42 C
  2. Incubate reaction from step 1 with Cas13 protein, guide RNA, and reporter molecule for 30 min at 37 C
  3. Dip the test strip into reaction from step 2, and result should appear within five minutes.

Further details which researchers and laboratories can follow (including guide RNA sequences), can be found in the .pdf protocol, which is available here and has been submitted to bioRxiv. The protocol will be updated as the team continues experiments in parallel and in partnership with those around the world seeking to address this outbreak. The researchers will continue to update this page with the most advanced solutions.

Necessary plasmids are available through the Zhang Lab Addgene repository, and other materials are commercially available. Details for how to obtain these materials are described in the protocol.

What’s next

The SHERLOCK diagnostic system has demonstrated success in other settings. The research team hopes the protocol is a useful step towards creating a system for detecting COVID-19 in patient samples using a simple readout. Further optimization, production, testing, and verification are still needed. Any diagnostic would need to follow all local regulations, best practices, and validation before it could become of actual clinical use. The researchers will continue to release and share protocol updates, and welcome updates from the community.

Organizations in any country interested in further developing and deploying this system for COVID-19 response can freely use the scientific instructions provided here and can email sherlock@broadinstitute.org for further free support, including guidance on developing a starter kit with the Cas13 protein, guide RNA, reporter molecule, and isothermal amplification primers.

Acknowledgments: The research team wishes to acknowledge support from the NIH (1R01- MH110049 and 1DP1-HL141201 grants); the Howard Hughes Medical Institute; McGovern Institute for Brain Research at MIT; the Poitras Center for Affective Disorders Research at MIT; Open Philanthropy Project; James and Patricia Poitras; and Robert Metcalfe.

Declaration of conflicts of interest: F.Z., O.O.A., and J.S.G. are inventors on patents related to Cas13, SHERLOCK, and CRISPR diagnostics, and are co-founders, scientific advisors, and hold equity interests in Sherlock Biosciences, Inc.

 

CRISPR makes several Discovery of the Decade lists

by Sabbi Lall |

As we reach milestones in time, it’s common to look back and review what we learned. A number of media outlets, including National Geographic, NPR, The Hill, Popular Mechanics, Smithsonian Magazine, Nature, Mental Floss, CNBC, and others, recognized the profound impact of genome editing, adding CRISPR to their discovery of the decade lists.

“In 2013, [CRISPR] was used for genome editing in a eukaryotic cell, forever altering the course of biotechnology and, ultimately our relationship with our DNA.”
— Popular Mechanics

It’s rare for a molecular system to become a household name, but in less than a decade, CRISPR has done just that. McGovern Investigator Feng Zhang played a key role in leveraging CRISPR, an immune system found originally in prokaryotic – bacterial and archaeal – cells, into a broadly customizable toolbox for genomic manipulation in eukaryotic (animal and plant) cells. CRISPR allows scientists to easily and quickly make changes to genomes, has revolutionized the biomedical sciences, and has major implications for control of infectious disease, agriculture, and treatment of genetic disorders.

Shrinking CRISPR tools

by Sabbi Lall |

Before CRISPR gene-editing tools can be used to treat brain disorders, scientists must find safe ways to deliver the tools to the brain. One promising method involves harnessing viruses that are benign, and replacing non-essential genetic cargo with therapeutic CRISPR tools. But there is limited room for additional tools in a vector already stuffed with essential gear.

Squeezing all the tools that are needed to edit the genome into a single delivery vector is a challenge. Soumya Kannan is addressing this capacity problem in Feng Zhang’s lab with fellow graduate student Han Altae-Tran, by developing smaller CRISPR tools that can be more easily packaged into viral vectors for delivery. She is focused on RNA editors, members of the Cas13 family that can fix small mutations in RNA without making changes to the genome itself.

“The limitation is that RNA editors are large. At this point though, we know that editing works, we understand the mechanism by which it works, and there’s feasible packaging in AAV. We’re now trying to shrink systems such as RESCUE and REPAIR so that they fit into the packaging for delivery.”

One of many avenues the Zhang lab has taken to tool-finding in the past is to explore biodiversity for new versions of tools, and this is an approach that intrigues Soumya.

“Metagenomics projects are literally sequencing life from the Antarctic ice cores to hot sea vents. It fascinates me that the CRISPR tools of ancient organisms and those that live in extreme conditions.”

Researchers continue to search these troves of sequencing data for new tools.

 

CRISPR: From toolkit to therapy

by Sabbi Lall |

Think of the human body as a community of cells with specialized roles. Each cell carries the same blueprint, an array of genes comprising the genome, but different cell types have unique functions — immune cells fight invading bacteria, while neurons transmit information.

But when something goes awry, the specialization of these cells becomes a challenge for treatment. For example, neurons lack active cell repair systems required for promising gene editing techniques like CRISPR.

Can current gene editing tools be modified to work in neurons? Can we reach neurons without impacting healthy cells nearby? McGovern Institute researchers are trying to answer these questions by developing gene editing tools and delivery systems that can target — and repair — faulty brain cells.

Expanding the toolkit

Feng Zhang with folded arms in lab
McGovern Investigator Feng Zhang in his lab.

Natural CRISPR systems help bacteria fend off would-be attackers. Our first glimpse of the impact of such systems was the use of CRISPR-Cas9 to edit human cells.

“Harnessing Cas9 was a major game-changer in the life sciences,” explains Feng Zhang, an investigator at the McGovern Institute and the James and Patricia Poitras Professor of Neuroscience at MIT. “But Cas9 is just one flavor of one kind of bacterial defense system — there is a treasure trove of natural systems that may have enormous potential, just waiting to be unlocked.”

By finding and optimizing new molecular tools, the Zhang lab and others have developed CRISPR tools that can now potentially target neurons and fix diverse mutation types, bringing gene therapy within reach.

Precise in space and time

A single letter change to a gene can be devastating. These genes may function only briefly during development, so a temporary “fix” during this window could be beneficial. For such cases, the Zhang lab and others have engineered tools that target short-lived RNAs. These molecules act as messengers, carrying information from DNA to be converted into functional factors in the cell.

“RNA editing is powerful from an ethical and safety standpoint,” explains Soumya Kannan, a graduate student in the Zhang lab working on these tools. “By targeting RNA molecules, which are only present for a short time, we can avoid permanent changes to the genetic material, and we can make these changes in any type of cell.”

Soumya Kannan in the lab
Graduate student Soumya Kannan is developing smaller CRISPR tools that can be more easily packaged into viral vectors for delivery. Photo: Caitlin Cunningham

Zhang’s team has developed twin RNA-editing tools, REPAIR and RESCUE, which can fix single RNA bases by bringing together a base editor with the CRISPR protein Cas13. These RNA-editing tools can be used in neurons because they do not rely on cellular machinery to make the targeted changes. They also have the potential to tackle a wide array of diseases in other tissue types.

CAST addition

If a gene is severely disrupted, more radical help may be needed: insertion of a normal gene. For this situation, Zhang’s lab recently identified CRISPR-associated transposases (CASTs) from cyanobacteria. CASTs combine Cas12k, which is targeted by a guide RNA to a precise genome location, with an enzyme that can insert gene-sized pieces of DNA.

“With traditional CRISPR you can make simple changes, similar to changing a few letters or words in a Word document. The new system can ‘copy and paste’ entire genes.” – Alim Ladha

Transposases were originally identified as enzymes that help rogue genes “jump” from one place to another in the genome. CAST uses a similar activity to insert entire genes self-sufficiently without help from the target cell so, like REPAIR and RESCUE, it can potentially be used in neurons.

“Our initial work was to fully characterize how this new system works, and test whether it can actually insert genes,” explains Alim Ladha, a graduate fellow in the Tan-Yang Center for Autism Research, who worked on CAST with Jonathan Strecker, a postdoctoral fellow in the Zhang lab.

The goal is now to use CAST to precisely target neurons and other specific cell types affected by disease.

Toward delivery

As the gene-editing toolbox expands, McGovern labs are working on precise delivery systems.Adeno-associated virus (AAV) is an FDA-approved virus for delivering genes, but has limited room to carry the necessary cargo — CRISPR machinery plus templates — to fix genes.

To tackle this problem, McGovern Investigators Guoping Feng and Feng Zhang are working on reducing the cargo needed for therapy. In addition, the Zhang, Gootenberg and Abudayyeh labs are working on methods to precisely deliver the therapeutic packages to neurons, such as new tissue-specific viruses that can carry bigger payloads. Finally, entirely new modalities for delivery are being explored in the effort to develop gene therapy to a point where it can be safely delivered to patients.

“Cas9 has been a very useful tool for the life sciences,” says Zhang. “And it’ll be exciting to see continued progress with the broadening toolkit and delivery systems, as we make further progress toward safe gene therapies.

McGovern scientists named STAT Wunderkinds

by Sabbi Lall |

McGovern researchers Sam Rodriques and Jonathan Strecker have been named to the class of 2019 STAT wunderkinds. This group of 22 researchers was selected from a national pool of hundreds of nominees, and aims to recognize trail-blazing scientists that are on the cusp of launching their careers but not yet fully independent.

“We were thrilled to receive this news,” said Robert Desimone, director of the McGovern Institute. “It’s great to see the remarkable progress being made by young scientists in McGovern labs be recognized in this way.”

Finding context

Sam Rodriques works in Ed Boyden’s lab at the McGovern Institute, where he develops new technologies that enable researchers to understand the behaviors of cells within their native spatial and temporal context.

“Psychiatric disease is a huge problem, but only a handful of first-in-class drugs for psychiatric diseases approved since the 1960s,” explains Rodriques, also affiliated with the MIT Media Lab and Broad Institute. “Coming up with novel cures is going to require new ways to generate hypotheses about the biological processes that underpin disease.”

Rodriques also works on several technologies within the Boyden lab, including preserving spatial information in molecular mapping technologies, finding ways of following neural connectivity in the brain, and Implosion Fabrication, or “Imp Fab.” This nanofabrication technology allows objects to be evenly shrunk to the nanoscale and has a wide range of potential applications, including building new miniature devices for examining neural function.

“I was very surprised, not expecting it at all!” explains Rodriques when asked about becoming a STAT Wunderkind, “I’m sure that all of the hundreds of applicants are very accomplished scientists, and so to be chosen like this is really an honor.”

New tools for gene editing

Jonathan Strecker is currently a postdoc working in Feng Zhang’s lab, and associated with both the McGovern Institute and Broad Institute. While CRISPR-Cas9 continues to have a profound effect and huge potential for research and biomedical, and agricultural applications, the ability to move entire genes into specific target locations remained out reach.

“Genome editing with CRISPR-Cas enzymes typically involves cutting and disrupting genes, or making certain base edits,” explains Strecker, “however, inserting large pieces of DNA is still hard to accomplish.”

As a postdoctoral researcher in the lab of CRISPR pioneer Feng Zhang, Strecker led research that showed how large sequences could be inserted into a genome at a given location.

“Nature often has interesting solutions to these problems and we were fortunate to identify and characterize a remarkable CRISPR system from cyanobacteria that functions as a programmable transposase.”

Importantly, the system he discovered, called CAST, doesn’t require cellular machinery to insert DNA. This is important as it means that CAST could work in many cell types, including those that have stopped dividing such as neurons, something that is being pursued.

By finding new sources of inspiration, be it nature or art, both Rodriques and Strecker join a stellar line up of young investigators being recognized for creativity and innovation.

 

New CRISPR platform expands RNA editing capabilities

by Sabbi Lall |

CRISPR-based tools have revolutionized our ability to target disease-linked genetic mutations. CRISPR technology comprises a growing family of tools that can manipulate genes and their expression, including by targeting DNA with the enzymes Cas9 and Cas12 and targeting RNA with the enzyme Cas13. This collection offers different strategies for tackling mutations. Targeting disease-linked mutations in RNA, which is relatively short-lived, would avoid making permanent changes to the genome. In addition, some cell types, such as neurons, are difficult to edit using CRISPR/Cas9-mediated editing, and new strategies are needed to treat devastating diseases that affect the brain.

McGovern Institute Investigator and Broad Institute of MIT and Harvard core member Feng Zhang and his team have now developed one such strategy, called RESCUE (RNA Editing for Specific C to U Exchange), described in the journal Science.

Zhang and his team, including first co-authors Omar Abudayyeh and Jonathan Gootenberg (both now McGovern Fellows), made use of a deactivated Cas13 to guide RESCUE to targeted cytosine bases on RNA transcripts, and used a novel, evolved, programmable enzyme to convert unwanted cytosine into uridine — thereby directing a change in the RNA instructions. RESCUE builds on REPAIR, a technology developed by Zhang’s team that changes adenine bases into inosine in RNA.

RESCUE significantly expands the landscape that CRISPR tools can target to include modifiable positions in proteins, such as phosphorylation sites. Such sites act as on/off switches for protein activity and are notably found in signaling molecules and cancer-linked pathways.

“To treat the diversity of genetic changes that cause disease, we need an array of precise technologies to choose from. By developing this new enzyme and combining it with the programmability and precision of CRISPR, we were able to fill a critical gap in the toolbox,” says Zhang, the James and Patricia Poitras Professor of Neuroscience at MIT. Zhang also has appointments in MIT’s departments of Brain and Cognitive Sciences and Biological Engineering.

Expanding the reach of RNA editing to new targets

The previously developed REPAIR platform used the RNA-targeting CRISPR/Cas13 to direct the active domain of an RNA editor, ADAR2, to specific RNA transcripts where it could convert the nucleotide base adenine to inosine, or letters A to I. Zhang and colleagues took the REPAIR fusion, and evolved it in the lab until it could change cytosine to uridine, or C to U.

RESCUE can be guided to any RNA of choice, then perform a C-to-U edit through the evolved ADAR2 component of the platform. The team took the new platform into human cells, showing that they could target natural RNAs in the cell as well as 24 clinically relevant mutations in synthetic RNAs. They then further optimized RESCUE to reduce off-target editing, while minimally disrupting on-target editing.

New targets in sight

Expanded targeting by RESCUE means that sites regulating activity and function of many proteins through post-translational modifications, such as phosphorylation, glycosylation, and methylation can now be more readily targeted for editing.

A major advantage of RNA editing is its reversibility, in contrast to changes made at the DNA level, which are permanent. Thus, RESCUE could be deployed transiently in situations where a modification may be desirable temporarily, but not permanently. To demonstrate this, the team showed that in human cells, RESCUE can target specific sites in the RNA encoding β-catenin, that are known to be phosphorylated on the protein product, leading to a temporary increase in β-catenin activation and cell growth. If such a change was made permanently, it could predispose cells to uncontrolled cell growth and cancer, but by using RESCUE, transient cell growth could potentially stimulate wound healing in response to acute injuries.

The researchers also targeted a pathogenic gene variant, APOE4.  The APOE4 allele has consistently emerged as a genetic risk factor for the development of late-onset Alzheimer’s Disease. Isoform APOE4 differs from APOE2, which is not a risk factor, by just two differences (both C in APOE4 vs. U in APOE2). Zhang and colleagues introduced the risk-associated APOE4 RNA into cells, and showed that RESCUE can convert its signature C’s to an APOE2 sequence, essentially converting a risk to a non-risk variant.

To facilitate additional work that will push RESCUE toward the clinic as well as enable researchers to use RESCUE as a tool to better understand disease-causing mutations, the Zhang lab plans to share the RESCUE system broadly, as they have with previously developed CRISPR tools. The technology will be freely available for academic research through the non-profit plasmid repository Addgene. Additional information can be found on the Zhang lab’s webpage.

Support for the study was provided by The Phillips Family; J. and P. Poitras; the Poitras Center for Psychiatric Disorders Research; Hock E. Tan and K. Lisa Yang Center for Autism Research.; Robert Metcalfe; David Cheng; a NIH F30 NRSA 1F30-CA210382 to Omar Abudayyeh. F.Z. is a New York Stem Cell Foundation–Robertson Investigator. F.Z. is supported by NIH grants (1R01-HG009761, 1R01-222 MH110049, and 1DP1-HL141201); the Howard Hughes Medical Institute; the New York Stem Cell Foundation and G. Harold and Leila Mathers Foundations.