Discovery by Doudna Lab & Collaborators Should Help Improve Accuracy of CRISPR-Cas9 Gene Editing

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. The protein domain the researchers identified as a master controller of DNA cutting is an obvious target for re-engineering to improve accuracy even further, the researchers say. 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. One strategy to achieve improved accuracy is to create mutations in the governing protein domain, called REC3, and see which ones improve accuracy without impacting the efficiency of on-target cutting. "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. As an extension of this work, one could perform a more unbiased mutagenesis within REC3 than the targeted mutations we have made," said co-first author Janice Chen, a graduate student in the lab of Dr. Jennifer Doudna, who co-invented the CRISPR-Cas9 gene-editing tool. Co-first authors Chen, Yavuz Dagdas, and Benjamin Kleinstiver, and their colleagues at UC Berkeley, Massachusetts General Hospital, and Harvard University reported their results online on September 20, 2017 in Nature. The article is titled “Enhanced Proofreading Governs CRISPR–Cas9 Targeting Accuracy.”
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