Division of Infectious Diseases, Department of Medicine, Columbia University Irving Medical Center, 630 West 168 Street, New York, NY 10032, USA.
STAR Protoc. 2021 Mar 6;2(1):100373. doi: 10.1016/j.xpro.2021.100373. eCollection 2021 Mar 19.
Multi-drug resistant (MDR) remains an urgent public health threat. While whole-genome sequencing has helped identify genetic changes underlying resistance, functional validation remains difficult due to a lack of genetic manipulation systems for MDR . CRISPR-Cas9 has revolutionized molecular biology, but its use was only recently adapted in bacteria by overcoming the lack of genetic repair systems. We describe a CRISPR-Cas9/lambda recombineering system utilizing a zeocin resistance cassette allowing efficient and versatile genetic manipulation of . For complete details on the use and execution of this protocol, please refer to McConville et al. (2020).
多药耐药（MDR）仍然是一个紧迫的公共卫生威胁。虽然全基因组测序有助于确定耐药性的遗传变化，但由于缺乏针对 MDR 的遗传操作系统，功能验证仍然很困难。CRISPR-Cas9 彻底改变了分子生物学，但最近通过克服缺乏遗传修复系统才在细菌中得以应用。我们描述了一个利用博来霉素抗性盒的 CRISPR-Cas9/λ重组酶系统，允许高效且多功能的遗传操作。有关此方案的使用和执行的完整详细信息，请参阅 McConville 等人（2020 年）。
