Development of the CRISPR-Cas9 gene-editing system has given rise to a new era of gene editing with wide applications in biology, medicine, agriculture, and other fields. However, the overexpression of Cas9 nuclease causes off-target effects and may trigger an immune response in vivo. Therefore, we constructed a self-restricting CRISPR-Cas9 system, where the target gene sequence corresponding to the guide RNA (gRNA) is inserted on either end of the Cas9 promoter. When double-strand breaks (DSBs) are induced in the target gene sequence, the Cas9 promoter is cut off and transcription ceases. With this system, expression of Cas9 protein at 60 h after transfection is only 10% that of the wild-type system, with about 70% promoter deletion efficiency. The target site editing efficiency and homologous recombination efficiency of the self-restricting system remain at about 50% and 30%, respectively, while the frequency of off-target indel formation decreased by 76.7%. Further, the number of indel types was also reduced from 13 to 2. Because this system does not include additional gRNA sequences, the possibility of introducing new off-target mutations is decreased. Importantly, this system is composed of a single plasmid, which could potentially be easily introduced in vivo using a viral vector or nanoparticles.

CRISPR-Cas9基因编辑系统的开发带来了基因编辑的新时代，其在生物学，医学，农业和其他领域的广泛应用。但是，Cas9核酸酶的过表达引起脱靶效应，并可能在体内触发免疫反应。因此，我们构建了一个自我限制的C​​RISPR-Cas9系统，其中与向导RNA（gRNA）相对应的目标基因序列插入到Cas9启动子的任一端。当在目标基因序列中诱导双链断裂（DSB）时，Cas9启动子被切断，转录停止。使用该系统，转染后60 h Cas9蛋白的表达仅为野生型系统的10％，启动子缺失效率约为70％。自限制性系统的目标位点编辑效率和同源重组效率分别保持在约50％和30％，而脱靶插入缺失形成的频率降低了76.7％。此外，插入缺失类型的数量也从13个减少到2个。由于该系统不包括其他gRNA序列，因此引入新的脱靶突变的可能性降低了。重要的是，该系统由单个质粒组成，可以使用病毒载体或纳米颗粒将其容易地体内导入。