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Incorporation of bridged nucleic acids into CRISPR RNAs improves Cas9 endonuclease specificity.

ABSTRACT: 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.

SUBMITTER: Cromwell CR

PROVIDER: S-EPMC5899152 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature

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Incorporation of bridged nucleic acids into CRISPR RNAs improves Cas9 endonuclease specificity.

Cromwell Christopher R CR Sung Keewon K Park Jinho J Krysler Amanda R AR Jovel Juan J Kim Seong Keun SK Hubbard Basil P BP

Nature communications 20180413 1

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'-BNA<sup>NC</sup>[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 B ...[more]

PMID: 29654299

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