Directed evolution of CRISPR-Cas9 to increase its specificity

Nat Commun. 2018 Aug 6;9(1):3048. doi: 10.1038/s41467-018-05477-x.


The use of CRISPR-Cas9 as a therapeutic reagent is hampered by its off-target effects. Although rationally designed S. pyogenes Cas9 (SpCas9) variants that display higher specificities than the wild-type SpCas9 protein are available, these attenuated Cas9 variants are often poorly efficient in human cells. Here, we develop a directed evolution approach in E. coli to obtain Sniper-Cas9, which shows high specificities without killing on-target activities in human cells. Unlike other engineered Cas9 variants, Sniper-Cas9 shows WT-level on-target activities with extended or truncated sgRNAs with further reduced off-target activities and works well in a preassembled ribonucleoprotein (RNP) format to allow DNA-free genome editing.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • CRISPR-Cas Systems*
  • DNA / genetics
  • Directed Molecular Evolution*
  • Escherichia coli / genetics
  • Gene Editing
  • HEK293 Cells
  • Humans
  • Plasmids / metabolism
  • RNA, Guide / genetics
  • Recombinant Proteins / chemistry
  • Ribonucleoproteins / chemistry
  • Substrate Specificity


  • RNA, Guide
  • Recombinant Proteins
  • Ribonucleoproteins
  • DNA