High-fidelity CRISPR-Cas9 Nucleases With No Detectable Genome-Wide Off-Target Effects

Nature. 2016 Jan 28;529(7587):490-5. doi: 10.1038/nature16526. Epub 2016 Jan 6.

Abstract

CRISPR-Cas9 nucleases are widely used for genome editing but can induce unwanted off-target mutations. Existing strategies for reducing genome-wide off-target effects of the widely used Streptococcus pyogenes Cas9 (SpCas9) are imperfect, possessing only partial or unproven efficacies and other limitations that constrain their use. Here we describe SpCas9-HF1, a high-fidelity variant harbouring alterations designed to reduce non-specific DNA contacts. SpCas9-HF1 retains on-target activities comparable to wild-type SpCas9 with >85% of single-guide RNAs (sgRNAs) tested in human cells. Notably, with sgRNAs targeted to standard non-repetitive sequences, SpCas9-HF1 rendered all or nearly all off-target events undetectable by genome-wide break capture and targeted sequencing methods. Even for atypical, repetitive target sites, the vast majority of off-target mutations induced by wild-type SpCas9 were not detected with SpCas9-HF1. With its exceptional precision, SpCas9-HF1 provides an alternative to wild-type SpCas9 for research and therapeutic applications. More broadly, our results suggest a general strategy for optimizing genome-wide specificities of other CRISPR-RNA-guided nucleases.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • CRISPR-Associated Proteins / genetics*
  • CRISPR-Associated Proteins / metabolism*
  • CRISPR-Cas Systems / physiology*
  • Clustered Regularly Interspaced Short Palindromic Repeats / genetics*
  • DNA / genetics
  • DNA / metabolism
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism
  • Endonucleases / genetics
  • Endonucleases / metabolism*
  • Genetic Engineering*
  • Genome, Human / genetics*
  • Humans
  • Mutation
  • Protein Binding
  • RNA / genetics
  • Reproducibility of Results
  • Sequence Analysis, DNA
  • Streptococcus pyogenes / enzymology
  • Streptococcus pyogenes / genetics
  • Substrate Specificity

Substances

  • CRISPR-Associated Proteins
  • DNA-Binding Proteins
  • RNA
  • DNA
  • Endonucleases

Associated data

  • SRA/SRP066862