Engineered Campylobacter jejuni Cas9 variant with enhanced activity and broader targeting range

Commun Biol. 2022 Mar 8;5(1):211. doi: 10.1038/s42003-022-03149-7.

Abstract

The RNA-guided DNA endonuclease Cas9 is a versatile genome-editing tool. However, the molecular weight of the commonly used Streptococcus pyogenes Cas9 is relatively large. Consequently, its gene cannot be efficiently packaged into an adeno-associated virus vector, thereby limiting its applications for therapeutic genome editing. Here, we biochemically characterized the compact Cas9 from Campylobacter jejuni (CjCas9) and found that CjCas9 has a previously unrecognized preference for the N3VRYAC protospacer adjacent motif. We thus rationally engineered a CjCas9 variant (enCjCas9), which exhibits enhanced cleavage activity and a broader targeting range both in vitro and in human cells, as compared with CjCas9. Furthermore, a nickase version of enCjCas9, but not CjCas9, fused with a cytosine deaminase mediated C-to-T conversions in human cells. Overall, our findings expand the CRISPR-Cas toolbox for therapeutic genome engineering.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • CRISPR-Cas Systems*
  • Campylobacter jejuni* / genetics
  • Gene Editing
  • Humans