High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects

Research output: Journal Publications and Reviews (RGC: 21, 22, 62)21_Publication in refereed journal

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Author(s)

  • Benjamin P. Kleinstiver
  • Vikram Pattanayak
  • Michelle S. Prew
  • Shengdar Q. Tsai
  • Nhu T. Nguyen
  • J. Keith Joung

Related Research Unit(s)

Detail(s)

Original languageEnglish
Pages (from-to)490-495
Journal / PublicationNature
Volume529
Issue number7587
StatePublished - 28 Jan 2016

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.

Citation Format(s)

High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects. / Kleinstiver, Benjamin P.; Pattanayak, Vikram; Prew, Michelle S.; Tsai, Shengdar Q.; Nguyen, Nhu T.; Zheng, Zongli; Joung, J. Keith.

In: Nature, Vol. 529, No. 7587, 28.01.2016, p. 490-495.

Research output: Journal Publications and Reviews (RGC: 21, 22, 62)21_Publication in refereed journal