Department of Laboratory Medicine and Pathology, University of Washington, Seattle, WA, USA.
Methods Mol Biol. 2022;2479:135-157. doi: 10.1007/978-1-0716-2233-9_10.
Recombineering has proven to be an extraordinarily powerful and versatile approach for the modification of bacterial genomes, but has historically not been possible in the important opportunistic pathogen Staphylococcus aureus. After evaluating the activity of various recombinases in S. aureus, we developed methods for recombineering in that organism using synthetic, single-stranded DNA oligonucleotides. This approach can be coupled to CRISPR/Cas9-mediated lethal counterselection in order to improve the efficiency with which recombinant S. aureus are recovered, which is especially useful in instances where mutants lack a selectable phenotype. These methods provide a rapid, scalable, precise, and inexpensive means to engineer point mutations, variable-length deletions, and short insertions into the S. aureus genome.
同源重组已被证明是一种非常强大且用途广泛的方法，可用于修饰细菌基因组，但在重要的机会性病原体金黄色葡萄球菌中，这种方法在历史上一直不可行。在评估了各种重组酶在金黄色葡萄球菌中的活性后，我们开发了使用合成单链 DNA 寡核苷酸进行该生物体同源重组的方法。这种方法可以与 CRISPR/Cas9 介导的致死性负选择相结合，以提高重组金黄色葡萄球菌的回收效率，这在突变体缺乏可选择表型的情况下特别有用。这些方法为在金黄色葡萄球菌基因组中进行点突变、可变长度缺失和短插入提供了一种快速、可扩展、精确且廉价的工程手段。
