An efficient and versatile CRISPR-Cas9 system for genetic manipulation of multi-drug resistant .

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).