DOI: 10.1101/496711Dec 17, 2018Paper

Native CRISPR-Cas mediated in situ genome editing reveals extensive resistance synergy in the clinical multidrug resistant Pseudomonas aeruginosa

BioRxiv : the Preprint Server for Biology
Zeling XuAixin Yan

Abstract

Antimicrobial resistance (AMR) is imposing a global public health threat. Despite its importance, characterizing drug resistance directly in the clinical isolates of resistant pathogens is frequently hindered by the lack of genome editing tools in these "non-model" strains. Pseudomonas aeruginosa is both a prototypical multidrug resistant (MDR) pathogen and a model species for CRISPR-Cas research. In this study, we report the successful development of a simple and efficient one-plasmid mediated, one-step genome editing approach in a paradigmatic MDR strain PA154197 by exploiting its native type I-F CRISPR-Cas system. The technique is readily applicable in two additional type I-F CRISPR-containing, clinical and environmental P. aeruginosa isolates. A two-step In-Del strategy involving insertion and subsequent deletion of a tag nearby the desired editing site is further developed to edit the genomic locus lacking an effective PAM (protospacer adjacent motif) or within an essential gene, which principally allows any non-lethal genomic manipulation in the strains. With these powerful techniques, the resistant determinants of PA154197 were delineated in its native genetic background. Moreover, relative contributions and extensive sy...Continue Reading

Related Concepts

Chloramphenicol
Drug Resistance
Gene Deletion
Genes
Genome
Laboratory
Plasmids
Pseudomonas aeruginosa
Research
Trimethoprim

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