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• How specific is CRISPR-Cas9 really?

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         description:Off-target DNA cleavage is a paramount concern when applying CRISPR-Cas9 gene-editing technology to functional genetics and human therapeutic applications. Here, we show that incorporation of next-generation bridged nucleic acids (2′,4′-BNANC[N-Me]) as well as locked nucleic acids (LNA) at specific locations in CRISPR-RNAs (crRNAs) broadly reduces off-target DNA cleavage by Cas9 in vitro and in cells by several orders of magnitude. Using single-molecule FRET experiments we show that BNANC incorporation slows Cas9 kinetics and improves specificity by inducing a highly dynamic crRNA–DNA duplex for off-target sequences, which shortens dwell time in the cleavage-competent, “zipped” conformation. In addition to describing a robust technique for improving the precision of CRISPR/Cas9-based gene editing, this study illuminates an application of synthetic nucleic acids. Minimizing off-target effects is an important concern for therapeutic applications of CRISPR-Cas9. Here, the authors show that incorporating bridged or locked nucleic acids into crRNA improves editing kinetics and reduces off-target cleavage.
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      headline:Incorporation of bridged nucleic acids into CRISPR RNAs improves Cas9 endonuclease specificity
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