Abstract
Protein evolution has emerged as a crucial tool for generating proteins with novel characteristics. A key step in protein evolution is the mutagenesis of protein-coding DNA. Error-prone PCR (epPCR) is a frequently used technique, but its low mutation efficiency often requires multiple rounds of mutagenesis, which can be time-consuming. To address this, we developed a novel DNA mutagenesis strategy termed deaminase-driven random mutation (DRM). DRM utilizes the engineered cytidine deaminase A3A-RL and the engineered adenosine deaminase ABE8e to introduce a broad spectrum of mutations, including C-to-T, G-to-A, A-to-G, and T-to-C, in both the protein-coding strand and the complementary strand. This approach enables the generation of a multitude of DNA mutation types within a single round of mutagenesis, resulting in a higher DNA mutagenic capability than epPCR. The results show that the DRM strategy exhibits a 14.6-fold higher DNA mutation frequency and produces a 27.7-fold greater diversity of mutation types compared to epPCR, enabling a more comprehensive exploration of the genetic landscape. This enhanced mutagenic capability increases the chances of discovering novel and useful mutants. With its ability to produce high-quality DNA products and the superior protein mutant generation capacity, DRM is an attractive tool for researchers seeking to engineer new proteins or improve existing ones.