Compact 9dBEs Enable Efficient and Precise Genome Editing in Mammalian Cells and In Vivo

Abstract

Despite the promise of DNA base editors for diverse genome editing applications, their utility remains constrained by off-target effects, which are exacerbated by short spacers in miniature systems and the large size of Cas9-derived editors, which impedes adeno-associated virus (AAV) delivery. Here, guided by structural insights into a compact Cas9d nuclease from Deltaproteobacteria, we developed an efficient Cas9d system (Cas9dUltra) through gRNA and protein engineering, and further developed its base editors (9dBEs). Cas9dUltra and 9dBEs enabled efficient and precise genome editing in human cells. Notably, 9dCBE induced premature termination codons in 89% of mouse pups by microinjection, facilitating robust disease modeling. Furthermore, a single AAV delivering 9dCBE achieved efficient Pcsk9 editing and a concomitant reduction in serum LDL-C levels in mice. Collectively, this study establishes a series of compact, potent genome editing tools poised to advance biological and biomedical translational research.