BACKGROUND CRISPR/Cas9 systems have been repurposed as canonical genome editing tools in a variety of species, but no application for the model strain Rhodobacter sphaeroides 2.4.1 was unveiled. RESULTS Here we showed two kinds of programmable base editing systems, cytosine base editors (CBEs) and adenine base editors (ABEs), generated by fusing endonuclease Cas9 variant to cytosine deaminase PmCDA1 or heterodimer adenine deaminase TadA-TadA*, respectively. Using CBEs, we were able to obtain C-to-T mutation of single and double targets following the first induction step, with the efficiency of up to 97% and 43%; while the second induction step was needed in the case of triple target, with the screening rate of 47%. Using ABEs, we were only able to gain A-to-G mutation of single target after the second induction step, with the screening rate of 30%. Additionally, we performed a knockout analysis to identify the genes responsible for coenzyme Q10 biosynthesis and found that ubiF, ubiA, ubiG, and ubiX to be the most crucial ones. CONCLUSIONS Together, CBEs and ABEs serve as alternative methods for genetic manipulation in Rhodobacter sphaeroides and will shed light on the fundamental research of other bacteria that are hard to be directly edited by Cas9-sgRNA.

中文翻译：

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CRISPR / Cas9-脱氨酶可在球形红球菌2.4.1中实现强大的碱基编辑。

背景技术CRISPR / Cas9系统已被重新用作多种物种的规范基因组编辑工具，但尚未公开球形红球菌2.4.1的模型菌株应用。结果在这里，我们展示了两种可编程的碱基编辑系统，分别是胞嘧啶碱基编辑器（CBEs）和腺嘌呤碱基编辑器（ABEs），它们是通过将核酸内切酶Cas9变体与胞嘧啶脱氨酶PmCDA1或异二聚腺嘌呤脱氨酶TadA-TadA *融合而产生的。使用CBE，我们能够在第一步诱导后获得单靶和双靶的C-T突变，效率高达97％和43％；而三重靶标则需要第二步诱导，筛选率为47％。使用ABE，我们只能在第二个诱导步骤后获得单个靶标的A至G突变，筛查率为30％。此外，我们进行了基因敲除分析以鉴定负责辅酶Q10生物合成的基因，并发现ubiF，ubiA，ubiG和ubiX是最关键的基因。结论CBEs和ABEs一起作为球形球形红细菌基因操作的替代方法，将为其他难以被Cas9-sgRNA直接编辑的细菌提供基础研究。