CRISPR/Cas9-Based Counterselection Boosts Recombineering Efficiency in Pseudomonas putida

TLDR: The results presented here upgrade the engineering possibilities of the genome of this environmental bacterium (and possibly other Gram-negatives) to obtain modifications that are otherwise cumbersome to generate.

Abstract: While adoption of single-stranded (ss) DNA recombineering techniques has greatly eased genetic design of the platform strain Pseudomonas putida KT2440, available methods still produce the desired modifications/deletions at low frequencies. This makes isolation of mutants that do not display selectable or conspicuous phenotypes considerably difficult. To overcome this limitation, we have merged ssDNA recombineering with CRISPR/Cas9 technology in this bacterium for efficient killing of unmodified cells and thus non-phenotypic selection of bacteria bearing the mutations of interest. After incorporating the system into standardized pSEVA plasmids we tested its functional efficiency by targeting different types of changes that ranged from single nucleotide substitutions to one-gene deletions—to even the removal a large flagellar cluster (∼69 kb). Simultaneous introduction of two independent gene deletions was tested as well. In all cases, directing the crRNA/Cas9 complexes towards non-modified, wild-type genomic sequences boosted dramatically the appearance of the mutants at stake in the absence of any phenotypic selection. The results presented here upgrade the engineering possibilities of the genome of this environmental bacterium (and possibly other Gram-negatives) to obtain rare modifications that are otherwise cumbersome to generate.