Host-Microbe Interactomics, Animal Sciences, Wageningen University, 6708 WD Wageningen, The Netherlands.
ACS Synth Biol. 2023 Sep 15;12(9):2546-2560. doi: 10.1021/acssynbio.3c00110. Epub 2023 Aug 21.
is an important zoonotic pathogen that causes severe invasive disease in pigs and humans. Current methods for genome engineering of rely on the insertion of antibiotic resistance markers, which is time-consuming and labor-intensive and does not allow the precise introduction of small genomic mutations. Here we developed a system for CRISPR-based genome editing in , utilizing linear DNA fragments for homologous recombination (HR) and a plasmid-based negative selection system for bacteria not edited by HR. To enable the use of this system in other bacteria, we engineered a broad-host-range replicon in the CRISPR plasmid. We demonstrated the utility of this system to rapidly introduce multiple gene deletions in successive rounds of genome editing and to make precise nucleotide changes in essential genes. Furthermore, we characterized a mechanism by which can escape killing by a targeted Cas9-sgRNA complex in the absence of HR. A characteristic of this new mechanism is the presence of very slow-growing colonies in a persister-like state that may allow for DNA repair or the introduction of mutations, alleviating Cas9 pressure. This does not impact the utility of CRISPR-based genome editing because the escape colonies are easily distinguished from genetically edited clones due to their small colony size. Our CRISPR-based editing system is a valuable addition to the genetic toolbox for engineering of , as it accelerates the process of mutant construction and simplifies the removal of antibiotic markers between successive rounds of genome editing.
是一种重要的人畜共患病病原体，可导致猪和人类的严重侵袭性疾病。目前对 的基因组工程方法依赖于抗生素抗性标记的插入，这既耗时又费力，并且不允许精确引入小的基因组突变。在这里，我们开发了一种基于 CRISPR 的 基因组编辑系统，利用线性 DNA 片段进行同源重组（HR），并使用基于质粒的负筛选系统来筛选未经过 HR 编辑的细菌。为了使该系统能够在其他细菌中使用，我们在 CRISPR 质粒中构建了一个广谱宿主复制子。我们证明了该系统在连续几轮基因组编辑中快速引入多个基因缺失以及在必需基因中进行精确核苷酸改变的用途。此外，我们还研究了 逃避靶向 Cas9-sgRNA 复合物杀伤的机制，而这种逃避机制的特征是存在处于类似持续状态的生长缓慢的菌落，这可能允许进行 DNA 修复或引入突变，从而减轻 Cas9 的压力。由于逃逸菌落由于其较小的菌落大小而容易与经过基因编辑的克隆区分开来，因此这并不影响基于 CRISPR 的基因组编辑的实用性。我们的基于 CRISPR 的编辑系统是对 工程遗传工具包的重要补充，因为它加速了突变构建的过程，并简化了在连续几轮基因组编辑之间去除抗生素标记的过程。
