Genome Editing in Mouse Embryo Using the CRISPR/Cas12i3 System.

The CRISPR/Cas system is a sizable family that is currently a popular and efficient gene editing tool. Cas12i3, as a member of the Type V-I family, has the characteristics of recognizing T-rich PAM sequences and being guided by shorter crRNA and has higher gene editing efficiency than Cas9 in rice. However, as a potential tool in accelerating the breeding process, the application of Cas12i3 in mammalian embryos has not yet been reported. Our study systematically evaluated the feasibility of applying CRISPR/Cas12i3 to gene editing in mouse embryos, with the core pluripotency regulator gene Nanog as the target. We successfully constructed a Nanog loss-of-function mouse embryo model using CRISPR/Cas12i3. At the targeted Nanog locus, its editing efficiency exceeded that of the Cas9 system under matched experimental conditions; no off-target phenomenon was detected. Moreover, the Cas12i3 system exhibited no side effect on mouse embryo development and proliferation of blastocyst cells. Finally, we obtained healthy chimeric gene-edited offspring by optimizing the concentration of the Cas12i3 mixture. These results confirm the feasibility and safety of CRISPR/Cas12i3 for gene editing in mammals, which provides a reliable tool for one-step generation of gene-edited animals for applications in biology, medical research, and large livestock breeding.