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

While adoption of single‐stranded 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...

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Bibliographic Details
Published in:Biotechnology journal 2018-05, Vol.13 (5), p.e1700161-n/a
Main Authors: Aparicio, Tomás, de Lorenzo, Víctor, Martínez‐García, Esteban
Format: Article
Language:English
Subjects:
CRISPR/Cas9
crRNA
escapers
genome engineering
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ssDNA recombineering
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Summary:While adoption of single‐stranded 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, the authors 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 the authors tested its functional efficiency by targeting different types of changes that ranged from single nucleotide substitutions to one‐gene deletions—to even the removal of the large flagellar cluster (≈69 kb). Simultaneous introduction of two independent gene deletions was tested as well. In all cases, directing the crRNA/Cas9 complexes toward 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 modifications that are otherwise cumbersome to generate. Some strains of Pseudomonas putida have been developed as chassis for engineering a suite of new properties of biotechnological interest. Optimization of such properties often requires a large number of genomic edits which are fastidious to implement with the technologies available thus far. ssDNA recombineering along with counterselection of wild‐type genomic sequences with Cas9/RNA complexes ease such genetic manipulations in this bacterium to an unprecedented degree.
ISSN:1860-6768
1860-7314
DOI:10.1002/biot.201700161