A highly specific SpCas9 variant is identified by in vivo screening in yeast

Nat Biotechnol. 2018 Mar;36(3):265-271. doi: 10.1038/nbt.4066. Epub 2018 Jan 29.


Despite the utility of CRISPR-Cas9 nucleases for genome editing, the potential for off-target activity limits their application, especially for therapeutic purposes. We developed a yeast-based assay to identify optimized Streptococcus pyogenes Cas9 (SpCas9) variants that enables simultaneous evaluation of on- and off-target activity. We screened a library of SpCas9 variants carrying random mutations in the REC3 domain and identified mutations that increased editing accuracy while maintaining editing efficiency. We combined four beneficial mutations to generate evoCas9, a variant that has fidelity exceeding both wild-type (79-fold improvement) and rationally designed Cas9 variants (fourfold average improvement), while maintaining near wild-type on-target editing efficiency (90% median residual activity). Evaluating evoCas9 on endogenous genomic loci, we demonstrated a substantially improved specificity and observed no off-target sites for four of the eight single guide RNAs (sgRNAs) tested. Finally, we showed that following long-term expression (40 d), evoCas9 strongly limited the nonspecific cleavage of a difficult-to-discriminate off-target site and fully abrogated the cleavage of two additional off-target sites.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • CRISPR-Associated Protein 9 / genetics*
  • CRISPR-Cas Systems / genetics*
  • Gene Editing*
  • Mutation
  • RNA, Guide / genetics*
  • Streptococcus pyogenes / enzymology
  • Streptococcus pyogenes / genetics
  • Substrate Specificity


  • RNA, Guide
  • CRISPR-Associated Protein 9