Base editing enables duplex point mutagenesis in Clostridium autoethanogenum at the price of numerous off-target mutations.
Base editing enables duplex point mutagenesis in Clostridium autoethanogenum at the price of numerous off-target mutations.
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DOI：
10.3389/fbioe.2023.1211197
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发表时间：
2023
影响因子：
5.7
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碱基编辑器是最近衍生自化脓链霉菌的Cas9核酸酶的多重基因编辑工具。它们可以靶向和修饰基因组中的单个核苷酸，而不诱导DNA螺旋的双链断裂（DSB）。因此，它们对于缺乏有效的DSB修复途径如同源重组（HR）或非同源末端连接（NHEJ）的微生物的工程改造具有巨大的潜力。然而，碱基编辑器在原核生物中的应用报道甚少，其优缺点也没有系统的报道。在这里，我们使用碱基编辑器Target-AID和Target-AID-NG将无义突变引入工业相关革兰氏阳性细菌自产乙醇梭菌的四个不同编码序列中。虽然可以使用多种多重策略同时编辑多达两个基因座，但大多数菌落表现出混合的基因型，并且大多数可用的原型间隔区在靶向编辑窗口内导致不期望的突变。此外，通过对所得菌株的基因组测序检测到15个脱靶突变，其中7个单核苷酸多态性（SNP）在与靶向原型间隔区具有一定相似性的基因座中或附近，1个15 nt重复，和1个12 kb缺失，其去除了尿嘧啶DNA糖基化酶（UDG），一种被认为是碱基编辑诱变的障碍的关键DNA修复酶。还示出了通过利用tRNA成熟机制来处理原核单向导RNA阵列的策略。
Base editors are recent multiplex gene editing tools derived from the Cas9 nuclease of Streptomyces pyogenes. They can target and modify a single nucleotide in the genome without inducing double-strand breaks (DSB) of the DNA helix. As such, they hold great potential for the engineering of microbes that lack effective DSB repair pathways such as homologous recombination (HR) or non-homologous end-joining (NHEJ). However, few applications of base editors have been reported in prokaryotes to date, and their advantages and drawbacks have not been systematically reported. Here, we used the base editors Target-AID and Target-AID-NG to introduce nonsense mutations into four different coding sequences of the industrially relevant Gram-positive bacterium Clostridium autoethanogenum. While up to two loci could be edited simultaneously using a variety of multiplexing strategies, most colonies exhibited mixed genotypes and most available protospacers led to undesired mutations within the targeted editing window. Additionally, fifteen off-target mutations were detected by sequencing the genome of the resulting strain, among them seven single-nucleotide polymorphisms (SNP) in or near loci bearing some similarity with the targeted protospacers, one 15 nt duplication, and one 12 kb deletion which removed uracil DNA glycosylase (UDG), a key DNA repair enzyme thought to be an obstacle to base editing mutagenesis. A strategy to process prokaryotic single-guide RNA arrays by exploiting tRNA maturation mechanisms is also illustrated.