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Editing RNA one letter at a time
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 October 25, 2017 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 nucleosides [Nucleosides are the basic building blocks of nucleic acids: ribonucleic acid (RNA) and deoxyriboneculeic acid (DNA)] 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," said Zhang, a core institute member at the Broad Institute and investigator at the McGovern Institute for Brain Research 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 which are 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 cases of focal epilepsy, Duchenne muscular dystrophy, and Parkinson's disease,for example. 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," explained co-first author David Cox, a graduate student in Zhang's lab.
To create REPAIR, researchers systematically looked for the CRISPR-Cas13 enzyme family as potential "editor" candidates (unlike Cas9, the Cas13 proteins target and cut RNA).
They selected an enzyme called PspCas13b taken from the Prevotella bacteria, it was the most effective at inactivating RNA. The team engineered a deactivated variation of PspCas13b that still binds to specific RNA stretches, but without the "scissor-like" technique. The deactivated PspCas13b then fused to a protein called ADAR2, changing the nucleoside adenosine to inosine in RNAs.
In REPAIR, the deactivated Cas13b enzyme looks for a target sequence on RNA, and the ADAR2 element performs the nucleoside conversion without cutting the transcript or relying on any of the cell's native machinery.
The team further modified the CRISPR-Cas13 editing system to improve specificity and reduce detectable off-target edits from 18,385 to only 20 in the whole transcriptome [the sum total of all the messenger RNA molecules expressed from the genes of an organism].
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.
To demonstrate REPAIR's therapeutic potential, the team replicated the disease-causing mutations behind Fanconi anemia and X-linked renal diabetes insipidus, introducing them into human cells, and then 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 research team is also working on additional tools for other types of nucleoside conversions. "There's immense natural diversity in these enzymes," said 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, plan 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.
Nucleic acid editing holds promise for treating genetic disease, particularly at the RNA level, where disease-relevant sequences can be rescued to yield functional protein products. Type VI CRISPR-Cas systems contain the programmable single-effector RNA-guided RNases Cas13. Here, we profile Type VI systems to engineer a Cas13 ortholog capable of robust knockdown and demonstrate RNA editing by using catalytically-inactive Cas13 (dCas13) to direct adenosine to inosine deaminase activity by ADAR2 to transcripts in mammalian cells. This system, referred to as RNA Editing for Programmable A to I Replacement (REPAIR), which has no strict sequence constraints, can be used to edit full-length transcripts containing pathogenic mutations. We further engineer this system to create a high specificity variant and minimize the system to facilitate viral delivery. REPAIR presents a promising RNA editing platform with broad applicability for research, therapeutics, and biotechnology.
Authors: David B. T. Cox, Jonathan S. Gootenberg, Omar O. Abudayyeh, Brian Franklin, Max J. Kellner, Julia Joung, Feng Zhang
This research was funded in part by the National Institutes of Health, grants 1R01-HG009761, 1R01-MH110049, and 1DP1-HL141201.
About the Broad Institute of MIT and Harvard
Broad Institute of MIT and Harvard was launched in 2004 to empower this generation of creative scientists to transform medicine. The Broad Institute seeks to describe all the molecular components of life and their connections; discover the molecular basis of major human diseases; develop effective new approaches to diagnostics and therapeutics; and disseminate discoveries, tools, methods, and data openly to the entire scientific community.
Founded by MIT, Harvard, Harvard-affiliated hospitals, and the visionary Los Angeles philanthropists Eli and Edythe L. Broad, the Broad Institute includes faculty, professional staff, and students from throughout the MIT and Harvard biomedical research communities and beyond, with collaborations spanning over a hundred private and public institutions in more than 40 countries worldwide. For further information about the Broad Institute, go to http://www.broadinstitute.org.
About the McGovern Institute
The McGovern Institute for Brain Research at MIT is led by a team of world-renowned neuroscientists committed to meeting two great challenges of modern science: understanding how the brain works and discovering new ways to prevent or treat brain disorders. The McGovern Institute was established in 2000 by Lore Harp McGovern and the late Patrick J. McGovern, with the goal of improving human welfare, communication and understanding through their support for neuroscience research. The director is Robert Desimone, formerly the head of intramural research at the National Institute of Mental Health.
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‘REPAIR’ system edits RNA, rather than DNA, substituting Adeosines for Inosines with the potential to treat diseases without permanently changing our genome. Image Credit: The Broad Institute