Genome editing with natural and engineered CjCas9 orthologs.

ABSTRACT: CjCas9 is one of the smallest CRISPR-associated (Cas9) nucleases for mammalian genome editing. However, it requires a long N₄RYAC (R = A or G; Y = C or T) protospacer-adjacent motif (PAM), limiting its DNA targeting scope. In this study, we investigated the PAMs of three CjCas9 orthologs, including Hsp1Cas9, Hsp2Cas9, and CcuCas9, by performing a GFP-activation assay. Interestingly, Hsp1Cas9 and CcuCas9 recognized unique N₄RAA and N₄CNA PAMs, respectively. We further generated an Hsp1Cas9-Hsp2Cas9 chimeric Cas9 (Hsp1-Hsp2Cas9), which recognized a simple N₄CY PAM. Genome-wide off-target analysis revealed that Hsp1-Hsp2Cas9 has very few off-targets compared to SpCas9. By analyzing the crystal structure of CjCas9, we identified eight mutations that

SUBMITTER: Gao S 

PROVIDER: S-EPMC10124074 | biostudies-literature | 2023 Apr

REPOSITORIES: biostudies-literature