Rationally engineered Cas9 nucleases with improved specificity

Science. 2016 Jan 1;351(6268):84-8. doi: 10.1126/science.aad5227. Epub 2015 Dec 1.

Abstract

The RNA-guided endonuclease Cas9 is a versatile genome-editing tool with a broad range of applications from therapeutics to functional annotation of genes. Cas9 creates double-strand breaks (DSBs) at targeted genomic loci complementary to a short RNA guide. However, Cas9 can cleave off-target sites that are not fully complementary to the guide, which poses a major challenge for genome editing. Here, we use structure-guided protein engineering to improve the specificity of Streptococcus pyogenes Cas9 (SpCas9). Using targeted deep sequencing and unbiased whole-genome off-target analysis to assess Cas9-mediated DNA cleavage in human cells, we demonstrate that "enhanced specificity" SpCas9 (eSpCas9) variants reduce off-target effects and maintain robust on-target cleavage. Thus, eSpCas9 could be broadly useful for genome-editing applications requiring a high level of specificity.

Publication types

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

MeSH terms

  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • DNA Cleavage*
  • Endonucleases / chemistry*
  • Endonucleases / genetics
  • Humans
  • Mutagenesis
  • Point Mutation
  • Protein Conformation
  • Protein Engineering*
  • RNA, Guide / genetics
  • Streptococcus pyogenes / enzymology*

Substances

  • Bacterial Proteins
  • RNA, Guide
  • Endonucleases