Phage-derived “recombineering” methods are utilized for bacterial genome editing. Recombineering results in a heterogeneous population of modified and unmodified chromosomes, and therefore selection methods, such as CRISPR-Cas9, are required to select for edited clones. Cells can evade CRISPR-Cas-induced cell death through recA-mediated induction of the SOS response. The SOS response increases RecA dependent repair as well as mutation rates through induction of the umuDC error prone polymerase. As a result, CRISPR-Cas selection is more efficient in recA mutants. We report an approach to inhibiting the SOS response and RecA activity through the expression of a mutant dominant negative form of RecA, which incorporates into wild type RecA filaments and inhibits activity. Using a plasmid-based system in which Cas9 and recA mutants are coexpressed, we can achieve increased efficiency and consistency of CRISPR-Cas9-mediated selection and recombineering in E. coli, while reducing the induction of the SOS response. To date, this approach has been shown to be independent of recA genotype and host strain lineage. Using this system, we demonstrate increased CRISPR-Cas selection efficacy with over 10 000 guides covering the E. coli chromosome. The use of dominant negative RecA or homologues may be of broad use in bacterial CRISPR-Cas-based genome editing where the SOS pathways are present.

中文翻译：

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通过瞬时抑制宿主RecA活性来管理SOS反应以增强大肠杆菌中基于CRISPR-Cas的重组

噬菌体衍生的“重组”方法用于细菌基因组编辑。重组导致修饰和未修饰染色体的异质群体，因此需要选择方法（例如CRISPR-Cas9）来选择可编辑的克隆。细胞可以通过recA介导的SOS反应诱导来逃避CRISPR-Cas诱导的细胞死亡。SOS响应通过umuDC易错聚合酶的诱导增加了RecA依赖的修复以及突变率。结果，CRISPR-Cas选择在recA中更有效突变体。我们报告了一种方法，通过表达一种突变的RecA显性负性负型表达来抑制SOS反应和RecA活性，该表达形式与野生型RecA细丝结合并抑制了活性。使用其中Cas9和recA突变体共表达的基于质粒的系统，我们可以在大肠杆菌中提高CRISPR-Cas9介导的选择和重组的效率和一致性，同时减少SOS反应的诱导。迄今为止，该方法已显示出与recA基因型和宿主菌株谱系无关。使用该系统，我们证明了超过10,000种覆盖大肠杆菌的指南提高了CRISPR-Cas选择效率染色体。显性阴性RecA或同源物的使用在存在SOS途径的细菌基于CRISPR-Cas的基因组编辑中可能具有广泛的用途。