Continuous directed evolution of a compact CjCas9 variant with broad PAM compatibility.

ABSTRACT: CRISPR-Cas9 genome engineering is a powerful technology for correcting genetic diseases. However, the targeting range of Cas9 proteins is limited by their requirement for a protospacer adjacent motif (PAM), and in vivo delivery is challenging due to their large size. Here, we use phage-assisted continuous directed evolution to broaden the PAM compatibility of Campylobacter jejuni Cas9 (CjCas9), the smallest Cas9 ortholog characterized to date. The identified variant, termed evoCjCas9, primarily recognizes N₄AH and N₅HA PAM sequences, which occur tenfold more frequently in the genome than the canonical N₃VRYAC PAM site. Moreover, evoCjCas9 exhibits higher nuclease activity than wild-type CjCas9 on canonical PAMs, with editing rates comparable to commonly use

SUBMITTER: Schmidheini L 

PROVIDER: S-EPMC7616171 | biostudies-literature | 2024 Mar

REPOSITORIES: biostudies-literature