Penewit Kelsi, Salipante Stephen J
Department of Laboratory Medicine, University of Washington, Seattle, WA, USA.
Methods Mol Biol. 2020;2050:127-143. doi: 10.1007/978-1-4939-9740-4_14.
Methods for the genetic manipulation of S. aureus have historically proven challenging, which has hindered experimental studies of this organism. We recently developed a system for recombineering and CRISPR/Cas9-mediated counterselection in S. aureus which utilizes commercially synthesized synthetic DNA oligonucleotides as substrates for introducing precise genomic modifications into the organism and for performing lethal counterselection of unedited cells. These techniques make it possible to scalably and inexpensively engineer desired genomic changes into laboratory or clinical S. aureus strains, using electroporation to introduce the effector plasmid vectors and oligonucleotides. Here we describe detailed protocols for performing genome editing of S. aureus in order to produce isogenic strains using this system and detail general principles which are broadly applicable across a range of organisms for which equivalent systems have been established.
历史证明，对金黄色葡萄球菌进行基因操作的方法具有挑战性，这阻碍了对该生物体的实验研究。我们最近开发了一种用于金黄色葡萄球菌重组工程和CRISPR/Cas9介导的反选择系统，该系统利用商业合成的DNA寡核苷酸作为底物，将精确的基因组修饰引入生物体，并对未编辑的细胞进行致死性反选择。这些技术使得利用电穿孔法导入效应质粒载体和寡核苷酸，能够以可扩展且经济的方式将所需的基因组变化引入实验室或临床金黄色葡萄球菌菌株中。在这里，我们描述了使用该系统对金黄色葡萄球菌进行基因组编辑以产生同基因菌株的详细方案，并详细阐述了广泛适用于已建立等效系统的一系列生物体的一般原则。
