In vivo genome editing with a novel Cj4Cas9

Natural CRISPR-Cas9 systems provide a rich resource for developing genome editing tools with diverse properties, including genome size, protospacer preference, and PAM specificity. In this study, we screened a panel of 11 Cas9 nucleases orthologous to CjCas9 using a GFP activation assay and identified seven active nucleases. Among these, Cj4Cas9 emerges as particularly noteworthy due to its compact genome size (985 amino acids) and unique PAM preference (5’-NNNGRY-3’). Cj4Cas9 demonstrates efficient disruption of the Tyr gene in mouse zygotes, resulting in an albino phenotype. Furthermore, when delivered via AAV8, Cj4Cas9 achieves efficient genome editing of the Pcsk9 gene in mouse liver, leading to reduced serum cholesterol and LDL-C levels. To enhance its utility, we engineered Cj4Cas9 for higher activity by introducing L58Y/D900K mutations, resulting in a variant termed enCj4Cas9. This variant exhibits a two-fold increase in nuclease activity compared to the wild-type Cj4Cas9 and recognizes a simplified N3GG PAM, considerably expanding its targeting scope. These findings highlight the potential of Cj4Cas9 and its high-activity variants for both fundamental research and therapeutic applications.