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

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

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.

CRISPR: From toolkit to therapy

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.

New CRISPR platform expands RNA editing capabilities

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.

Welcoming the first McGovern Fellows

We are delighted to kick off the new year by welcoming Omar Abuddayeh and Jonathan Gootenberg as the first members of our new McGovern Institute Fellows Program. The fellows program is a recently launched initiative that supports highly-talented and selected postdocs that are ready to initiate their own research program.

As McGovern Fellows, the pair will be given space, time, and support to help them follow scientific research directions of their own choosing. This provides an alternative to the traditional postdoctoral research route.

Abudayyeh and Gootenberg both defended their thesis in the fall of 2018, and graduated from the lab of Feng Zhang, who is the James and Patricia Poitras Professor of Neuroscience at MIT, a McGovern investigator and core member of the Broad Institute. During their time in the Zhang lab, Abudayyeh and Gootenberg worked on projects that sought and found new tools based on enzymes mined from bacterial CRISPR systems. Cas9 is the original programmable single-effector DNA-editing enzyme, and the new McGovern Fellows worked on teams that actively looked for CRISPR enzymes with properties distinct from and complementary to Cas9. In the course of their thesis work, they helped to identify RNA-guided RNA editing factors such as the Cas13 family. This work led to the development of the REPAIR system, which is capable of editing RNA, thus providing a CRISPR-based therapeutic avenue that is not based on permanent, heritable changes to the genome. In addition, they worked on a Cas13-based diagnostic system called SHERLOCK that can detect specific nucleic acid sequences. SHERLOCK is able to detect the presence of infectious agents such as Zika virus in an easily-deployable lateral flow format, similar to a pregnancy test.

We are excited to see the directions that the new McGovern Fellows take as they now arrive at the institute, and will keep you posted on scientific findings as they emerge from their labs.


What is CRISPR?

CRISPR (which stands for Clustered Regularly Interspaced Short Palindromic Repeats) is not actually a single entity, but shorthand for a set of bacterial systems that are found with a hallmarked arrangement in the bacterial genome.

When CRISPR is mentioned, most people are likely thinking of CRISPR-Cas9, now widely known for its capacity to be re-deployed to target sequences of interest in eukaryotic cells, including human cells. Cas9 can be programmed to target specific stretches of DNA, but other enzymes have since been discovered that are able to edit DNA, including Cpf1 and Cas12b. Other CRISPR enzymes, Cas13 family members, can be programmed to target RNA and even edit and change its sequence.

The common theme that makes CRISPR enzymes so powerful, is that scientists can supply them with a guide RNA for a chosen sequence. Since the guide RNA can pair very specifically with DNA, or for Cas13 family members, RNA, researchers can basically provide a given CRISPR enzyme with a way of homing in on any sequence of interest. Once a CRISPR protein finds its target, it can be used to edit that sequence, perhaps removing a disease-associated mutation.

In addition, CRISPR proteins have been engineered to modulate gene expression and even signal the presence of particular sequences, as in the case of the Cas13-based diagnostic, SHERLOCK.

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Omar Abudayyeh

Mining Bacteria for Tools

Omar Abudayyeh works on novel genome editing and gene delivery tools, and applying these tools towards the study of aging. While CRISPR is perhaps best known as a DNA editing tool, Abudayyeh’s team has discovered new CRISPR enzymes, such as the RNA-targeting CRISPR-Cas13, and adapted these enzymes for novel technologies, such as a simple and inexpensive tool to detect human disease. This technology, called SHERLOCK, can detect viruses, bacteria – even genetic signatures associated with cancer – in virtually any location. Abudayyeh and McGovern Fellow Jonathan Gootenberg continue to mine bacterial systems for new technologies to better enable gene therapy as well as applying these technologies towards studying mechanisms of aging.

Using next-generation single-cell sequencing and novel tools for perturbing cell states, Dr. Abudayyeh is determining, with unprecedented resolution, the cell types that arise in the aging brain and how mechanisms, such as senescence, drive detrimental processes in tissues. The ultimate goal is to use this information for building a detailed roadmap of aging circuits and to eventually reverse states of aging for regenerating tissues like the brain.

SHERLOCK: A CRISPR tool to detect disease

This animation depicts how Cas13 — a CRISPR-associated protein — may be adapted to detect human disease. This new diagnostic tool, called SHERLOCK, targets RNA (rather than DNA), and has the potential to transform research and global public health.


Researchers advance CRISPR-based tool for diagnosing disease

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


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

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

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

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

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

Clear results on a paper strip

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

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

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

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

Multiple targets and increased sensitivity

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

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

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

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

Researchers engineer CRISPR to edit single RNA letters in human cells

The Broad Institute and MIT scientists who first harnessed CRISPR for mammalian genome editing have engineered a new molecular system for efficiently editing RNA in human cells. RNA editing, which can alter gene products without making changes to the genome, has profound potential as a tool for both research and disease treatment.

In a paper published today in Science, senior author Feng Zhang and his team describe the new CRISPR-based system, called RNA Editing for Programmable A to I Replacement, or “REPAIR.” The system can change single RNA nucleotides in mammalian cells in a programmable and precise fashion. REPAIR has the ability to reverse disease-causing mutations at the RNA level, as well as other potential therapeutic and basic science applications.

“The ability to correct disease-causing mutations is one of the primary goals of genome editing,” says Zhang, a core institute member of the Broad Institute, an investigator at the McGovern Institute, and the James and Patricia Poitras ’63 Professor in Neuroscience and associate professor in the departments of Brain and Cognitive Sciences and Biological Engineering at MIT. “So far, we’ve gotten very good at inactivating genes, but actually recovering lost protein function is much more challenging. This new ability to edit RNA opens up more potential opportunities to recover that function and treat many diseases, in almost any kind of cell.”

REPAIR has the ability to target individual RNA letters, or nucleosides, switching adenosines to inosines (read as guanosines by the cell). These letters are involved in single-base changes known to regularly cause disease in humans. In human disease, a mutation from G to A is extremely common; these alterations have been implicated in, for example, cases of focal epilepsy, Duchenne muscular dystrophy, and Parkinson’s disease. REPAIR has the ability to reverse the impact of any pathogenic G-to-A mutation regardless of its surrounding nucleotide sequence, with the potential to operate in any cell type.

Unlike the permanent changes to the genome required for DNA editing, RNA editing offers a safer, more flexible way to make corrections in the cell. “REPAIR can fix mutations without tampering with the genome, and because RNA naturally degrades, it’s a potentially reversible fix,” explains co-first author David Cox, a graduate student in Zhang’s lab.

To create REPAIR, the researchers systematically profiled the CRISPR-Cas13 enzyme family for potential “editor” candidates (unlike Cas9, the Cas13 proteins target and cut RNA). They selected an enzyme from Prevotella bacteria, called PspCas13b, which was the most effective at inactivating RNA. The team engineered a deactivated variant of PspCas13b that still binds to specific stretches of RNA but lacks its “scissor-like” activity, and fused it to a protein called ADAR2, which changes the letters A to I in RNA transcripts.

In REPAIR, the deactivated Cas13b enzyme seeks out a target sequence of RNA, and the ADAR2 element performs the base conversion without cutting the transcript or relying on any of the cell’s native machinery.

The team further modified the editing system to improve its specificity, reducing detectable off-target edits from 18,385 to only 20 in the whole transcriptome. The upgraded incarnation, REPAIRv2, consistently achieved the desired edit in 20 to 40 percent — and up to 51 percent — of a targeted RNA without signs of significant off-target activity. “The success we had engineering this system is encouraging, and there are clear signs REPAIRv2 can be evolved even further for more robust activity while still maintaining specificity,” says Omar Abudayyeh, co-first author and a graduate student in Zhang’s lab. Cox and Abudayyeh are both students in the Harvard-MIT Program in Health Sciences and Technology.

To demonstrate REPAIR’s therapeutic potential, the team synthesized the pathogenic mutations that cause Fanconi anemia and X-linked nephrogenic diabetes insipidus, introduced them into human cells, and successfully corrected these mutations at the RNA level. To push the therapeutic prospects further, the team plans to improve REPAIRv2’s efficiency and to package it into a delivery system appropriate for introducing REPAIRv2 into specific tissues in animal models.

The researchers are also working on additional tools for other types of nucleotide conversions. “There’s immense natural diversity in these enzymes,” says co-first author Jonathan Gootenberg, a graduate student in both Zhang’s lab and the lab of Broad core institute member Aviv Regev. “We’re always looking to harness the power of nature to carry out these changes.”

Zhang, along with the Broad Institute and MIT, plans to share the REPAIR system widely. As with earlier CRISPR tools, the groups will make this technology freely available for academic research via the Zhang lab’s page on the plasmid-sharing website Addgene, through which the Zhang lab has already shared reagents more than 42,000 times with researchers at more than 2,200 labs in 61 countries, accelerating research around the world.

This research was funded, in part, by the National Institutes of Health and the Poitras Center for Affective Disorders Research.