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)