CRISPR/Cas system-guided plasmid mutagenesis without sequence restriction

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.