Genome Editing in Klebsiella pneumoniae Using CRISPR/Cas9 Technology

CRISPR/Cas9 systems have been widely adopted for genetic manipulation in diverse biological systems owing to the ease of use and high efficiency. We have recently developed a CRISPR/Cas9-based genome editing system (pCasKP-pSGKP) by coupling a CRISPR/Cas9 system with the lambda Red recombination system as well as a cytidine deaminase-mediated base editing system (pBECKP) in Klebsiella pneumoniae, enabling rapid, scarless, and efficient genetic manipulation in diverse K. pneumoniae strains. In this chapter, we introduce the detailed procedures of using these two tools for genome editing in K. pneumoniae.