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

Christopher R. Cromwell, Keewon Sung, Jinho Park, Amanda R. Krysler, Juan Jovel, Seong Keun Kim and Basil P. Hubbard ()
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Christopher R. Cromwell: University of Alberta
Keewon Sung: Seoul National University
Jinho Park: Seoul National University
Amanda R. Krysler: University of Alberta
Juan Jovel: University of Alberta
Seong Keun Kim: Seoul National University
Basil P. Hubbard: University of Alberta

Nature Communications, 2018, vol. 9, issue 1, 1-11

Abstract: 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.

Date: 2018
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DOI: 10.1038/s41467-018-03927-0

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