An efficient and versatile CRISPR-Cas9 system for genetic manipulation of multi-drug resistant <em>Klebsiella pneumoniae</em>

STAR Protoc. 2021 Mar 6;2(1):100373. doi: 10.1016/j.xpro.2021.100373. eCollection 2021 Mar 19.


Multi-drug resistant (MDR) Klebsiella pneumoniae 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 K. pneumoniae. 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 K. pneumoniae. For complete details on the use and execution of this protocol, please refer to McConville et al. (2020).

PMID:33733242 | PMC:PMC7941085 | DOI:10.1016/j.xpro.2021.100373