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Mutations Making CRISPR Hyper-Accurate Discovered

저자:   업로드:2017-09-25  조회수:

    The potential benefits of the CRISPR gene-editing system have been championed by a huge array of researchers, clinicians, and biotech companies. While the data in recent years supports that CRISPR/Cas9 is effective in altering genes to circumvent many disease states, the limitation of the system thus far has been the tendency of the Cas9 enzyme to cut at off-target sites.


    Now, scientists at the University of California, Berkeley and Massachusetts General Hospital have identified a key region within the Cas9 protein that governs how accurately CRISPR/Cas9 homes in on a target DNA sequence, and have tweaked it to produce a hyper-accurate gene editor with the lowest level of off-target cutting to date. Findings from the new study were published recently in Nature in an article entitled “Enhanced Proofreading Governs CRISPR–Cas9 Targeting Accuracy.”


    The research team identified a protein domain, called REC3, that acts as a master controller of DNA cutting—an obvious target for re-engineering to improve accuracy even further. This approach should help scientists customize variants of Cas9—the protein that binds and cuts DNA—to minimize the chance that CRISPR/Cas9 will edit DNA at the wrong place, a key consideration when doing gene therapy in humans.


    “We have found that even minor alterations in the REC3 domain of Cas9 affect the differential between on- and off-target editing, which suggests that this domain is an obvious candidate for in-depth mutagenesis to improve targeting specificity,” explained co-lead study investigator Janice Chen, a graduate student in the lab of Jennifer Doudna, Ph.D.—co-inventor of the CRISPR/Cas9 gene-editing tool. “As an extension of this work, one could perform a more unbiased mutagenesis within REC3 than the targeted mutations we have made."



    In the past several years, researchers have engineered highly accurate Cas9 proteins—an enhanced specificity one called eSpCas9(1.1) and a high-fidelity one called SpCas9-HF1— and Dr. Doudna and Ms. Chen sought to learn why they cut with higher specificity than the wild-type Cas9 protein from Streptococcus pyogenes used widely today.


    Currently, researchers using CRISPR/Cas9 create a single-guide RNA (sgRNA)—an RNA molecule that includes a chain of 20 ribonucleic acids that complements a specific 20-nucleic-acid DNA sequence they want to target—and attach it to Cas9. This guide RNA allows Cas9 to home in on the complementary DNA, bind to it, and cut the double-stranded helix. But the Cas9–sgRNA complex can also bind to DNA that doesn't exactly match, leading to undesirable off-target cutting.


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