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Protein evolution has significantly enhanced the development of life science. However, it is difficult to achieve  in vitro  evolution of some special proteins because of difficulties with heterologous expression, purification, and function detection. To achieve protein evolution via  in situ  mutation  in vivo , we developed a base editor by fusing nCas with a cytidine deaminase in  Bacillus subtilis  through genome integration. The base editor introduced a cytidine-to-thymidine mutation of approximately 100% across a 5 nt editable window, which was much higher than those of other base editors. The editable window was expanded to 8 nt by extending the length of sgRNA, and conversion efficiency could be regulated by changing culture conditions, which was suitable for constructing a mutant protein library efficiently  in vivo . As proof-of-concept, the Sec-translocase complex and bacitracin-resistance-related protein BceB were successfully evolved  in vivo  using the base editor. A Sec mutant with 3.6-fold translocation efficiency and the BceB mutants with different sensitivity to bacitracin were obtained. As the construction of the base editor does not rely on any additional or host-dependent factors, such base editors (BEs) may be readily constructed and applicable to a wide range of bacteria for protein evolution via  in situ  mutation.

Development of a base editor for protein evolution via in situ mutation in vivo