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

Abstract

<i>Klebsiella pneumoniae</i> is a promising industrial microorganism as well as a major human pathogen. The recent emergence of carbapenem-resistant <i>K. pneumoniae</i> has posed a serious threat to public health worldwide, emphasizing a dire need for novel therapeutic means against drug-resistant <i>K. pneumoniae</i> Despite the critical importance of genetics in bioengineering, physiology studies, and therapeutic-means development, genome editing, in particular, the highly desirable scarless genetic manipulation in <i>K. pneumoniae</i>, is often time-consuming and laborious. Here, we report a two-plasmid system, pCasKP-pSGKP, used for precise and iterative genome editing in <i>K. pneumoniae</i> By harnessing the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 genome cleavage system and the lambda Red recombination system, pCasKP-pSGKP enabled highly efficient genome editing in <i>K. pneumoniae</i> using a short repair template. Moreover, we developed a cytidine base-editing system, pBECKP, for precise C→T conversion in both the chromosomal and plasmid-borne genes by engineering the fusion of the cytidine deaminase APOBEC1 and a Cas9 nickase. By using both the pCasKP-pSGKP and the pBECKP tools, the <i>bla</i>_KPC-2 gene was confirmed to be the major factor that contributed to the carbapenem resistance of a hypermucoviscous carbapenem-resistant <i>K. pneumoniae</i> strain. The development of the two editing tools will significantly facilitate the genetic engineering of <i>K. pneumoniae</i><b>IMPORTANCE</b> 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 <i>K. pneumoniae</i> 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 <i>K. pneumoniae</i> strains. We applied both tools in dissecting the drug resistance mechanism of a hypermucoviscous carbapenem-resistant <i>K. pneumoniae</i> strain, elucidating that the <i>bla</i>_KPC-2 gene was the major factor that contributed to the carbapenem resistance of the hypermucoviscous carbapenem-resistant <i>K. pneumoniae</i> strain. Utilization of the two tools will dramatically accelerate a wide variety of investigations in diverse <i>K. pneumoniae</i> strains and relevant <i>Enterobacteriaceae</i> species, such as gene characterization, drug discovery, and metabolic engineering.