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.

中文翻译：

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通过体内原位突变开发蛋白质进化的碱基编辑器

蛋白质进化极大地促进了生命科学的发展。然而，由于异源表达、纯化和功能检测困难，一些特殊蛋白难以实现体外进化。为了通过体内原位突变实现蛋白质进化，我们开发了一种碱基编辑器，通过基因组整合将 nCas 与枯草芽孢杆菌中的胞苷脱氨酶融合。碱基编辑器在 5 nt 可编辑窗口中引入了大约 100% 的胞苷到胸苷突变，这远高于其他碱基编辑器。通过延长sgRNA的长度将可编辑窗口扩大到8 nt，并且可以通过改变培养条件来调节转化效率，适合在体内高效构建突变蛋白库。作为概念验证，Sec 转位酶复合物和杆菌肽抗性相关蛋白 BceB 使用碱基编辑器在体内成功进化。获得了具有3.6倍易位效率的Sec突变体和对杆菌肽具有不同敏感性的BceB突变体。由于碱基编辑器的构建不依赖于任何额外的或宿主依赖性因素，因此此类碱基编辑器 (BE) 可以很容易地构建并适用于通过原位突变进行蛋白质进化的广泛细菌。