Iterative engineering of a compact Cas9 ortholog for in vivo gene editing via single AAV delivery

ABSTRACT The development of compact and efficient CRISPR-Cas systems is crucial for biomedical and therapeutic genome editing, particularly in vivo applications based on viral delivery. Here, we performed a comparative functional screen of seven Cas9 orthologs to systematically evaluate their genome editing activity in mammalian cells. Among these, Cme2—a 1008 amino acid nuclease recognizing a 5′-NAGNGC PAM—emerged as a promising candidate based on its compact size and baseline editing activity. To overcome its limited native efficiency, we employed a dual engineering approach combining sgRNA scaffold optimization and rational protein mutagenesis. The resulting variant, en Cme2, exhibits markedly improved editing efficiency across multiple loci in both mouse and human cells while maintaining extremely high specificity and minimal off-target activity. Importantly, the small size of en Cme2 permits packaging of the complete system into a single rAAV vector, enabling efficient genome editing/HDR in in vivo tissues and mouse embryos, and facile generation of transgenic models. These results establish en Cme2 as a compact, precise, and AAV-compatible genome editing platform with broad applicability for in vivo research and therapeutic approaches, especially where high specificity is desirable.