Combination of ssDNA recombineering and CRISPR-Cas9 for Pseudomonas putida KT2440 genome editing.

Pseudomonas putida KT2440 is a Gram-negative, biosafety strain that plays important roles in environmental and biotechnological applications. Highly efficient genome editing strategy is essential to the elucidation of gene function and construction of metabolic engineered strains. Building on our previously established recombineering-mediated markerless and scarless P. putida KT2440 chromosomal gene deletion methods, herein we combined single-stranded DNA (ssDNA) recombineering and CRISPR-Cas9 technologies for P. putida KT2440 genome editing. Firstly, an inactive kanamycin resistance gene was knocked into the P. putida KT2440 chromosome. Then, based on kanamycin selection, recombinase gene selection, ssDNA recombineering condition optimization, and gRNA expression promoter selection were performed. A two-plasmid genome editing system was established; the first is a broad host range, RK2 replicon-based plasmid cloned with the tightly regulated redβ and cas9 genes; the second is a broad host range, pBBR1 replicon-based, sgRNA expression plasmid. Gene point mutations and gene deletions were carried out; the genome editing efficiency is as high as 100%. The method will expedite the P. putida KT2440 metabolic engineering and synthetic biology studies.