Zhao Fengjiao, Chen Feng, Yu Huahang, Fan Siyue, Bai Min, Xue Jing, Zhao Yue, Zuo Xiaolei, Fan Chunhai, Zhao Yongxi
Institute of Analytical Chemistry and Instrument for Life Science, The Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science and Technology, Xi'an Jiaotong University, Xi'an 710049, China.
School of Chemistry and Chemical Engineering, Frontiers Science Center for Transformative Molecules and National Center for Translational Medicine, Shanghai Jiao Tong University, Shanghai 200240, China.
Fundam Res. 2022 Jul 15;5(4):1481-1487. doi: 10.1016/j.fmre.2022.06.017. eCollection 2025 Jul.
Plasmid mutagenesis is an essential step to engineer protein variants with desired properties. Direct generation of mutations on plasmids is a promising method compared to PCR-based methods and subcloning, yet it suffers from the sequence restriction in the generation of single-stranded circular plasmids. Here we demonstrate the CRISPR/Cas system-guided plasmid mutagenesis, a method using gRNA/Cas9 nickase complex to generate single-stranded circular plasmids as polymerization templates for mutagenesis. This method can directly create user-defined mutation libraries on the plasmid. It offers broad sequence programmability and even covers methylated plasmids. We further combine this method with rational design to engineer genome-editing protein FnCpf1. The FnCpf1 variants with mutations in the PAM-binding groove were generated. Several variants expanded the PAM range and exhibited lower off-target effect, which can loosen PAM constraint and enhance target specificity for genome editing. This work presents an effective tool for plasmid mutagenesis and protein engineering studies.
质粒诱变是构建具有所需特性的蛋白质变体的关键步骤。与基于PCR的方法和亚克隆相比，在质粒上直接产生突变是一种很有前景的方法，但它在单链环状质粒的产生中存在序列限制。在此，我们展示了CRISPR/Cas系统引导的质粒诱变，这是一种使用gRNA/Cas9切口酶复合物产生单链环状质粒作为诱变聚合模板的方法。该方法可以直接在质粒上创建用户定义的突变文库。它具有广泛的序列可编程性，甚至可以处理甲基化质粒。我们进一步将该方法与合理设计相结合，对基因组编辑蛋白FnCpf1进行工程改造。产生了在PAM结合凹槽中具有突变的FnCpf1变体。几个变体扩大了PAM范围并表现出较低的脱靶效应，这可以放宽PAM限制并增强基因组编辑的靶标特异性。这项工作为质粒诱变和蛋白质工程研究提供了一个有效的工具。
