Abstract:Pseudomonas aeruginosa is one of the most common model bacterial species, and genomes of hundreds of strains of this species have been sequenced to date. However, currently there is only one available genome of an oceanic isolate. Here, we report two complete and six draft genome sequences of P. aeruginosa isolates from the open ocean.
“… Note: Concentration of kanamycin for selection is dependent on the kanamycin susceptibility of the P. aeruginosa isolates subjected to editing. For example, 800 μg/mL is used for selection of another clinical isolate PA150567 and 500 μg/mL for an environmental isolate Ocean-100 ( Kumagai et al., 2017 ). Incubate the plates at 37°C for 24 h. Figure 6 Prolonged Growth during Recovery of PA154197 Cells Electroporated with pEditing Resulted in Undesirable Cell Lysis Suspension of cells electroporated with the control plasmid pAY5211 or pEditing following recovery for 120 min.…”
Section: Step-by-step Methods Detailsmentioning
confidence: 99%
“…Note: Concentration of kanamycin for selection is dependent on the kanamycin susceptibility of the P. aeruginosa isolates subjected to editing. For example, 800 μg/mL is used for selection of another clinical isolate PA150567 and 500 μg/mL for an environmental isolate Ocean-100 ( Kumagai et al., 2017 ).…”
SUMMARY
Repurposing the broadly distributed native CRISPR-Cas systems in prokaryotes for genome editing is emerging as a new strategy for genetic manipulations. We recently reported the establishment of a single plasmid-mediated, one-step genome-editing technique in a multidrug-resistant genotype of the opportunistic pathogen
Pseudomonas aeruginosa
by harnessing its endogenous type I-F CRISPR-Cas system. The platform is readily applicable in additional type I-F CRISPR-containing clinical and environmental
P. aeruginosa
isolates. Herein, we provide the detailed protocol for the methodology.
For complete details on the establishment and exploitation of this protocol, please refer to
Xu et al. (2019)
.
“… Note: Concentration of kanamycin for selection is dependent on the kanamycin susceptibility of the P. aeruginosa isolates subjected to editing. For example, 800 μg/mL is used for selection of another clinical isolate PA150567 and 500 μg/mL for an environmental isolate Ocean-100 ( Kumagai et al., 2017 ). Incubate the plates at 37°C for 24 h. Figure 6 Prolonged Growth during Recovery of PA154197 Cells Electroporated with pEditing Resulted in Undesirable Cell Lysis Suspension of cells electroporated with the control plasmid pAY5211 or pEditing following recovery for 120 min.…”
Section: Step-by-step Methods Detailsmentioning
confidence: 99%
“…Note: Concentration of kanamycin for selection is dependent on the kanamycin susceptibility of the P. aeruginosa isolates subjected to editing. For example, 800 μg/mL is used for selection of another clinical isolate PA150567 and 500 μg/mL for an environmental isolate Ocean-100 ( Kumagai et al., 2017 ).…”
SUMMARY
Repurposing the broadly distributed native CRISPR-Cas systems in prokaryotes for genome editing is emerging as a new strategy for genetic manipulations. We recently reported the establishment of a single plasmid-mediated, one-step genome-editing technique in a multidrug-resistant genotype of the opportunistic pathogen
Pseudomonas aeruginosa
by harnessing its endogenous type I-F CRISPR-Cas system. The platform is readily applicable in additional type I-F CRISPR-containing clinical and environmental
P. aeruginosa
isolates. Herein, we provide the detailed protocol for the methodology.
For complete details on the establishment and exploitation of this protocol, please refer to
Xu et al. (2019)
.
“…To examine the applicability of the technique in other type I-F CRISPR-Cas containing P. aeruginosa strains, we set out to construct mexB deletion in a carbapenem resistant clinical strain PA150567 (accession number: LSQQ00000000) isolated from the Queen Mary Hospital, Hong Kong and an environmental strain Ocean-100 (accession number: NMRS00000000) isolated from the North Pacific Ocean [37]. As expected, transformation of the targeting plasmid pAY5233 led to DNA interference in the two strains, and the editing plasmid pAY5235 achieved the desired mexB deletion with comparable successful rate as in PA154197 (S3 Fig), confirming the general applicability of the developed editing system in clinically and environmentally isolated, “non-model” P. aeruginosa strains.…”
23Antimicrobial resistance (AMR) is imposing a global public health threat. Despite its 24 importance, resistance characterization in the native background of clinically isolated resistant 25 pathogens is frequently hindered by the lack of genome editing tools in these "non-model" 26 strains. Pseudomonas aeruginosa is both a prototypical multidrug resistant (MDR) pathogen 27 and a model species for understanding CRISPR-Cas functions. In this study, we report the 28 successful development of the first native type I-F CRISPR-Cas mediated, one-step genome 29 editing technique in a paradigmatic MDR strain PA154197. The technique is readily applicable 30 in additional type I-F CRISPR-containing, clinical/environmental P. aeruginosa isolates. A 31 two-step In-Del strategy is further developed to edit genomic locus lacking an effective PAM 32 (protospacer adjacent motif) or within an essential gene, which together principally allows any 33 type of non-lethal genomic manipulations in these strains. Exploiting these powerful 34 techniques, a series of reverse mutations are constructed and the key resistant determinants of 35 the MDR PA154197 are elucidated which include over-production of two multidrug efflux 36 pumps MexAB-OprM and MexEF-OprN, and a typical fluoroquinolone (FQ) resistance 37 mutation T83I in the drug target gene gyrA. Characterizing antimicrobial susceptibilities in 38 isogenic strains containing various combinations of single, double, or all three key resistance 39 determinants reveal that i) extensive synergy exists between the target mutation and over-40 production of efflux pumps, and between the two over-produced tripartite efflux pumps to 41 confer clinically significant FQ resistance; ii) while basal level MexAB-OprM confers 42 resistance only to penicillins, its over-production leads to substantial resistance to all 43 antipseudonmonal β-lactams and additional resistance to FQs; iii) despite the acquisition and 44 over-production of multiple resistant mutations, no obvious evolutionary trade-off of collateral 45 sensitivity is developed in PA154197. Together, these results provide new insights into 46 3 resistance development in clinical MDR P. aeruginosa strains and demonstrate the great 47 potentials of native CRISPR systems in AMR research. 48 49 Author Summary 50 Genome editing and manipulation can revolutionize the understanding, exploitation, and 51 control of microbial species. Despite the presence of well-established genetic manipulation 52 tools in various model strains, their applicability in the medically, environmentally, and 53 industrially important, "non-model" strains is often hampered owing to the vast diversity of 54 DNA homeostasis in these strains and the cytotoxicity of the heterologous CRISPR-Cas9/Cpf1 55 systems. Harnessing the native CRISPR-Cas systems broadly distributed in prokaryotes with 56 built-in genome targeting activity presents a promising and effective approach to resolve these 57 obstacles. We explored and exploited this methodology in the prototypical multi...
“…A series of antibiotic‐resistant genes were deleted in this genotype which enabled dissection of resistant mechanisms and development of a novel anti‐resistance strategy to eliminate MDR P. aeruginosa strains. The editing platform was also shown to be applicable in an environmental P. aeruginosa isolate Ocean‐100 (Kumagai et al, 2017) and another clinical isolate PA150567 (Xu et al, 2019).…”
Section: General Workflow Of Repurposing the Native Type I Crispr‐casmentioning
Summary
Genetic analysis is crucial to the understanding, exploitation, and control of microorganisms. The advent of CRISPR‐Cas‐based genome‐editing techniques, particularly those mediated by the single‐effector (Cas9 and Cas12a) class 2 CRISPR‐Cas systems, has revolutionized the genetics in model eukaryotic organisms. However, their applications in prokaryotes are rather limited, largely owing to the exceptional diversity of DNA homeostasis in microorganisms and severe cytotoxicity of overexpressing these nuclease proteins in certain genotypes. Remarkably, CRISPR‐Cas systems belonging to different classes and types are continuously identified in prokaryotic genomes and serve as a deep reservoir for expansion of the CRISPR‐based genetic toolkits. ~90% of the CRISPR‐Cas systems identified so far belong to the class 1 system which hinges on multi‐protein effector complexes for DNA interference. Harnessing these widespread native CRISPR‐Cas systems for ‘built‐in’ genome editing represents an emerging and powerful genetic tool in prokaryotes, especially in the genetically recalcitrant non‐model species and strains. In this progress review, we introduce the general workflow of this emerging editing platform and summarize its establishment in a growing number of prokaryotes by harnessing the most widespread, diverse type I CRISPR‐Cas systems present in their genomes. We also discuss the various factors affecting the success and efficiency of this editing platform and the corresponding solutions.
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