He Jiale, Liu Juan, Yue Yuan, Wang Lin, Liu Zhize, Xi Guangyin, An Lei, Tian Jianhui, Wang Yinjuan
Frontiers Science Center for Molecular Design Breeding (MOE), Key Laboratory of Animal Genetics, Breeding and Reproduction of the Ministry of Agriculture and Rural Affairs, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing 100193, China.
Int J Mol Sci. 2025 Mar 26;26(7):3036. doi: 10.3390/ijms26073036.
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 as the target. We successfully constructed a loss-of-function mouse embryo model using CRISPR/Cas12i3. At the targeted 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.
CRISPR/Cas系统是一个规模较大的家族，目前是一种流行且高效的基因编辑工具。Cas12i3作为V-I型家族的成员，具有识别富含T的PAM序列以及由较短的crRNA引导的特性，并且在水稻中具有比Cas9更高的基因编辑效率。然而，作为加速育种过程的一种潜在工具，Cas12i3在哺乳动物胚胎中的应用尚未见报道。我们的研究系统地评估了将CRISPR/Cas12i3应用于小鼠胚胎基因编辑的可行性，以核心多能性调节基因作为靶点。我们使用CRISPR/Cas12i3成功构建了一个功能缺失的小鼠胚胎模型。在靶向位点，其编辑效率在匹配的实验条件下超过了Cas9系统；未检测到脱靶现象。此外，Cas12i3系统对小鼠胚胎发育和囊胚细胞增殖没有副作用。最后，我们通过优化Cas12i3混合物的浓度获得了健康的嵌合基因编辑后代。这些结果证实了CRISPR/Cas12i3用于哺乳动物基因编辑的可行性和安全性，为一步生成用于生物学、医学研究和大型家畜育种的基因编辑动物提供了可靠工具。
