Conclusion
In this work, we established a targeted in vivo mutagenesis system by fusing different DNA-modifying enzymes, cytidine deaminase and T7 RNA polymerase. With the introduction of the DNA-modifying enzymes and the dual T7 promoters, our mutagenesis tools could raise the mutation frequency up to 5.13x10-3 and significantly expand the mutation spectrum. Besides transition mutations, our system could also efficiently generate transversion mutations, which are difficult to access by cytidine/adenosine-bearing evolutionary tools. Although our system still showed a slight bias toward generating C→T mutations, we hypothesized that this problem would be solved by employing more DNA-modifying enzymes and their combinations. Our mutagenesis tools are effective, flexible, and compatible with multiple evolutionary scenarios. Even without the selection pressure, our system functioned robustly and generated diverse mutations. In conclusion, our mutagenesis system could significantly increase the mutation frequency of target genes and generate mutations of different types, including transversion mutations, providing a powerful tool to accelerate the evolutionary process.

Conflict of interest

The authors declare that there is no conflict of interest.

Acknowledgments

This work was supported by the National Key Research and Development Program of China (2018YFA0900100) and Tianjin Fund for Distinguished Young Scholars (19JCJQJC63300)