In vivo genome editing with a novel Cj4Cas9.

Natural CRISPR-Cas9 systems provides diverse properties for genome editing, yet finding compact variants remains a priority. In this study, we screened a panel of 11 CjCas9 orthologous using a GFP activation assay and identified seven active nucleases. Among these, Cj4Cas9 stood out 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. Seeking to further expand 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 establish Cj4Cas9 and its engineered variants for fundamental research and therapeutic applications.