CRISPR-Cas9 and CRISPR-Assisted Cytidine Deaminase Enable Precise and Efficient Genome Editing in Klebsiella pneumoniae

Genetics is a key means to study bacterial physiology. However, the highly desirable scarless genetic manipulation is often time-consuming and laborious for the major human pathogen K. pneumoniae . We developed a CRISPR-Cas9-mediated genome-editing method and a cytidine base-editing system, enabling rapid, highly efficient, and iterative genome editing in both industrial and clinically isolated K. pneumoniae strains. We applied both tools in dissecting the drug resistance mechanism of a hypermucoviscous carbapenem-resistant K. pneumoniae strain, elucidating that the bla KPC-2 gene was the major factor that contributed to the carbapenem resistance of the hypermucoviscous carbapenem-resistant K. pneumoniae strain. Utilization of the two tools will dramatically accelerate a wide variety of investigations in diverse K. pneumoniae strains and relevant Enterobacteriaceae species, such as gene characterization, drug discovery, and metabolic engineering.