| Abstract |
CRISPR gene editing technologies have transformed functional genomics and biotechnology. Despite these advances, challenges such as limited delivery capacity and off-target activity continue to hinder their therapeutic translation. We developed a chimeric gene editing platform by fusing the compact, catalytically inactive Cas12m guiding module (GoCas12m) with the FokI nuclease domain. GoCas12m-FokI system integrates the programmable DNA-binding capability of Cas12m with the dimerization-dependent cleavage mechanism of FokI, enabling precise genome editing. Our engineered XTEN-fused GoCas12m-FokI editor exhibits robust activity on both surrogate reporters and endogenous human loci, achieving high-efficiency editing at clinically relevant targets-including CLTA1, HBB, AIFM1, and ABL with no detectable off-target activity at in silico-predicted sites, as confirmed by targeted deep sequencing. Notably, GoCas12m-FokI is nearly half the size of conventional Cas9- or Cas12a-based editors, facilitating delivery via adeno-associated virus and other cargo-limited vectors. This dual-guided editor showed comparable editing efficiency to previously reported FokI-dCas9 systems on endogenous loci, while possessing a different PAM requirement and domain orientation. By combining compact architecture, high specificity, and modular programmability, the GoCas12m-FokI editor offers a powerful alternative for therapeutic genome editing and a promising tool for in vivo gene therapy applications.
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