Precise genetic manipulation is vital to studying bacterial physiology, but is difficult to achieve in some bacterial species due to the weak intrinsic homologous recombination (HR) capacity and lack of a compatible exogenous HR system. Here we report the establishment of a rapid and efficient method for directly converting adenine to guanine in bacterial genomes using the fusion of an adenine deaminase and a Cas9 nickase. The method achieves the conversion of adenine to guanine via an enzymatic deamination reaction and a subsequent DNA replication process rather than HR, which is utilized in conventional bacterial genetic manipulation methods, thereby substantially simplifying the genome editing process. A systematic screening targeting the possibly editable adenine sites of cntBC, the importer of the staphylopine/metal complex in Staphylococcus aureus, pinpoints key residues for metal importation, demonstrating that application of the system would greatly facilitate the genomic engineering of bacteria.

中文翻译：

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使用Cas9-腺嘌呤-脱氨酶融合蛋白在细菌中进行可编程腺嘌呤脱氨。

精确的基因操作对于研究细菌生理至关重要，但由于固有的同源重组（HR）能力弱和缺乏兼容的外源HR系统，因此在某些细菌中很难实现。在这里，我们报道了使用腺嘌呤脱氨酶和Cas9切口酶的融合，在细菌基因组中将腺嘌呤直接转化为鸟嘌呤的快速有效方法的建立。该方法通过酶促脱氨反应和随后的DNA复制过程而不是HR来实现腺嘌呤向鸟嘌呤的转化，而HR是在常规细菌遗传操作方法中使用的，从而大大简化了基因组编辑过程。针对cntBC可能存在的腺嘌呤位点的系统筛选，