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

Nat Commun. 2018 Apr 13;9(1):1448. doi: 10.1038/s41467-018-03927-0.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • CRISPR-Cas Systems*
  • Cell Line, Tumor
  • Clustered Regularly Interspaced Short Palindromic Repeats*
  • DNA / chemistry*
  • DNA Cleavage
  • Endonucleases / metabolism
  • Escherichia coli
  • Fluorescence Resonance Energy Transfer
  • HeLa Cells
  • Humans
  • Polymorphism, Single Nucleotide
  • RNA / chemistry*
  • RNA, Guide, CRISPR-Cas Systems / genetics*
  • Sensitivity and Specificity
  • Streptococcus pyogenes

Substances

  • RNA, Guide, CRISPR-Cas Systems
  • RNA
  • DNA
  • Endonucleases