Acetogenic bacteria are rising in popularity as chassis microbes in biotechnology due to their capability of converting inorganic one-carbon (C1) gases to organic chemicals. To fully uncover the potential of acetogenic bacteria, synthetic-biology tools are imperative to either engineer designed functions or to interrogate the physiology. Here, we report a genome-editing tool at a one-nucleotide resolution, namely base editing, for acetogenic bacteria based on CRISPR-targeted deamination. This tool combines nuclease deactivated Cas9 with activation-induced cytidine deaminase to enable cytosine-to-thymine substitution without DNA cleavage, homology-directed repair, and donor DNA, which are generally the bottlenecks for applying conventional CRISPR-Cas systems in bacteria. We designed and validated a modularized base-editing tool in the model acetogenic bacterium Clostridium ljungdahlii. The editing principles were investigated, and an in-silico analysis revealed the capability of base editing across the genome. Moreover, genes related to acetate and ethanol production were disrupted individually by installing premature STOP codons to reprogram carbon flux towards improved acetate production. This resulted in engineered C. ljungdahlii strains with the desired phenotypes and stable genotypes. Our base-editing tool promotes the application and research in acetogenic bacteria and provides a blueprint to upgrade CRISPR-Cas-based genome editing in bacteria in general.

中文翻译：

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通过脱氨作用用CRISPR靶向碱基编辑对产乙酸细菌进行重编程

产乙酸细菌由于能够将无机一碳（C1）气体转化为有机化学物质，因此在生物技术中作为底盘微生物正在日益普及。为了充分挖掘产乙酸细菌的潜力，必须使用合成生物学工具来设计工程师设计的功能或询问生理学。在这里，我们报告了一种基于CRISPR靶向脱氨作用的产乙酸细菌的单核苷酸分辨率基因组编辑工具，即碱基编辑。该工具将核酸酶失活的Cas9与活化诱导的胞苷脱氨酶结合在一起，可实现胞嘧啶至胸腺嘧啶的取代而无需进行DNA切割，同源性定向修复和供体DNA，这通常是在细菌中应用常规CRISPR-Cas系统的瓶颈。杨氏梭菌。研究了编辑原理，并且进行了计算机分析，揭示了整个基因组中碱基编辑的能力。此外，通过安装过早的STOP密码子重新编程碳通量以提高乙酸盐的产生，可单独破坏与乙酸盐和乙醇的产生有关的基因。这产生了具有所需表型和稳定基因型的工程化的C.ljungdahlii菌株。我们的基础编辑工具促进了产乙酸细菌的应用和研究，并提供了一个蓝图，以升级总体上基于CRISPR-Cas的细菌基因组编辑。