Researcher: Omar Abudayyeh

Researchers engineer CRISPR to edit single RNA letters in human cells

Anne Trafton |

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.

Feng Zhang describes new system for genome engineering

Anne Trafton |

A team including the scientist who first harnessed the CRISPR-Cas9 system for mammalian genome editing has now identified a different CRISPR system with the potential for even simpler and more precise genome engineering.

In a study published today in Cell, Feng Zhang and his colleagues at the Broad Institute of MIT and Harvard and the McGovern Institute for Brain Research at MIT, with co-authors Eugene Koonin at the National Institutes of Health, Aviv Regev of the Broad Institute and the MIT Department of Biology, and John van der Oost at Wageningen University, describe the unexpected biological features of this new system and demonstrate that it can be engineered to edit the genomes of human cells.

“This has dramatic potential to advance genetic engineering,” says Eric Lander, director of the Broad Institute. “The paper not only reveals the function of a previously uncharacterized CRISPR system, but also shows that Cpf1 can be harnessed for human genome editing and has remarkable and powerful features. The Cpf1 system represents a new generation of genome editing technology.”

CRISPR sequences were first described in 1987, and their natural biological function was initially described in 2010 and 2011. The application of the CRISPR-Cas9 system for mammalian genome editing was first reported in 2013, by Zhang and separately by George Church at Harvard University.

In the new study, Zhang and his collaborators searched through hundreds of CRISPR systems in different types of bacteria, searching for enzymes with useful properties that could be engineered for use in human cells. Two promising candidates were the Cpf1 enzymes from bacterial species Acidaminococcus and Lachnospiraceae, which Zhang and his colleagues then showed can target genomic loci in human cells.

“We were thrilled to discover completely different CRISPR enzymes that can be harnessed for advancing research and human health,” says Zhang, the W.M. Keck Assistant Professor in Biomedical Engineering in MIT’s Department of Brain and Cognitive Sciences.

The newly described Cpf1 system differs in several important ways from the previously described Cas9, with significant implications for research and therapeutics, as well as for business and intellectual property:

  • First: In its natural form, the DNA-cutting enzyme Cas9 forms a complex with two small RNAs, both of which are required for the cutting activity. The Cpf1 system is simpler in that it requires only a single RNA. The Cpf1 enzyme is also smaller than the standard SpCas9, making it easier to deliver into cells and tissues.
  • Second, and perhaps most significantly: Cpf1 cuts DNA in a different manner than Cas9. When the Cas9 complex cuts DNA, it cuts both strands at the same place, leaving “blunt ends” that often undergo mutations as they are rejoined. With the Cpf1 complex the cuts in the two strands are offset, leaving short overhangs on the exposed ends. This is expected to help with precise insertion, allowing researchers to integrate a piece of DNA more efficiently and accurately.
  • Third: Cpf1 cuts far away from the recognition site, meaning that even if the targeted gene becomes mutated at the cut site, it can likely still be recut, allowing multiple opportunities for correct editing to occur.
  • Fourth: The Cpf1 system provides new flexibility in choosing target sites. Like Cas9, the Cpf1 complex must first attach to a short sequence known as a PAM, and targets must be chosen that are adjacent to naturally occurring PAM sequences. The Cpf1 complex recognizes very different PAM sequences from those of Cas9. This could be an advantage in targeting some genomes, such as in the malaria parasite as well as in humans.

“The unexpected properties of Cpf1 and more precise editing open the door to all sorts of applications, including in cancer research,” says Levi Garraway, an institute member of the Broad Institute, and the inaugural director of the Joint Center for Cancer Precision Medicine at the Dana-Farber Cancer Institute, Brigham and Women’s Hospital, and the Broad Institute. Garraway was not involved in the research.

An open approach to empower research

Zhang, along with the Broad Institute and MIT, plan to share the Cpf1 system widely. As with earlier Cas9 tools, these 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 Cas9 reagents more than 23,000 times with researchers worldwide to accelerate research. The Zhang lab also offers free online tools and resources for researchers through its website.

The Broad Institute and MIT plan to offer nonexclusive licenses to enable commercial tool and service providers to add this enzyme to their CRISPR pipeline and services, further ensuring availability of this new enzyme to empower research. These groups plan to offer licenses that best support rapid and safe development for appropriate and important therapeutic uses.

“We are committed to making the CRISPR-Cpf1 technology widely accessible,” Zhang says. “Our goal is to develop tools that can accelerate research and eventually lead to new therapeutic applications. We see much more to come, even beyond Cpf1 and Cas9, with other enzymes that may be repurposed for further genome editing advances.”