CRISPR Pseudomonas Aeruginosa Services | Gene Knockout, Knockin and Point Mutation, etc.

Pseudomonas aeruginosa is a common encapsulated, Gram-negative, rod-shaped bacterium that can cause disease in plants and animals, including humans. A species of considerable medical importance, P. aeruginosa is a multidrug resistant pathogen recognized for its ubiquity, its intrinsically advanced antibiotic resistance mechanisms, and its association with serious illnesses – hospital-acquired infections such as ventilator-associated pneumonia and various sepsis syndromes. This type of bacteria is found commonly in the environment, like in soil and in water. P. Aeruginosa is a gram-negative, aerobic rod bacterium of the Pseudomonadaceae family (a member of the Gammaproteobacteria). These years, P. aeruginosa are getting more popular in the field of CRISPR editing, which involves gene knockout, gene knock-in and point-mutation in the bacteria.

Applying CRISPR in P. aeruginosa Involving Insertion and Knockout of a Tag Gene

Pseudomonas aeruginosa is both a prototypical multidrug-resistant (MDR) pathogen and a model species for CRISPR-Cas research. The technique is readily applicable in two additional types I-F CRISPR-containing, clinical, and environmental P. aeruginosa isolates. A two-step In-Del strategy involving insertion and subsequent knockout 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. Among the three resistant mutations synergizing fluoroquinolones resistance, gyrA mutations elicit a greater resistance than drug efflux by MexAB-OprM or MexEF-OprN. These results advanced the understanding of the MDR development of clinical P. aeruginosa strains and demonstrated the great potentials of native CRISPR systems in AMR research. Despite the presence of well-established genetic manipulation tools in various model strains, their applicability in the medically, environmentally, and industrially significant, “non-model” strains is often hampered owing to the vast diversity of DNA homeostasis in these strains and the cytotoxicity of the heterologous CRISPR-Cas9/Cpf1 system. Harnessing the native CRISPR-Cas systems broadly distributed in prokaryotes with built-in genome targeting activity presents a promising and effective approach to resolve these obstacles. The successful development of the first type I-F CRISPR-mediated genome editing technique and its subsequent extension to additional clinical and environmental P. aeruginosa isolates opened a new avenue to the functional genomics of antimicrobial resistance in pathogens.

Silencing and Point Mutations in P. aeruginosa Helps Research in Bacterial Physiology, Drug Target Exploration, and Metabolic Engineering

Pseudomonas species exhibit significant biomedical, ecological, and industrial importance. Despite the extensive research and wide applications, genetic manipulation in Pseudomonas species, in particular in the major human pathogen Pseudomonas aeruginosa, remains a laborious endeavor. It is reported that a genome-editing method pCasPA/pACRISPR was developed by harnessing the CRISPR/Cas9 and the phage λ-Red recombination systems. The method allows for efficient and scarless genetic manipulation in P. aeruginosa. By engineering the fusion of the cytidine deaminase APOBEC1 and the Cas9 nickase, a base editing system pnCasPA-BEC was developed, which enables highly efficient gene inactivation and point mutations in a variety of Pseudomonas species, such as P. aeruginosa. Application of the two genome editing methods will dramatically accelerate a wide variety of investigations, such as bacterial physiology study, drug target exploration, and metabolic engineering.

The Rapid Construction of Gene Knockouts in P. aeruginosa with Base-pair Precision

Pseudomonas aeruginosa is a model organism for the study of quorum sensing, biofilm formation, and also leading cause of nosocomial infections in immune-compromised patients. As such P. aeruginosa is one of the most well-studied organisms in terms of its genetics. However, the construction of gene KOs and replacements in Pseudomonas aeruginosa is relatively time-consuming, requiring multiple steps including suicide vector construction, conjugation, inactivation with the insertion of antibiotic resistance cassettes and allelic exchange. Even employing Gateway recombineering techniques with direct transformation requires a minimum of two weeks. Hence, a rapid streamlined method was developed to create clean KO mutants in P. aeruginosa through direct transformation, eliminating the need for the creation of Gateway-compatible suicide vectors. In this method, upstream and downstream sequences of the gene/locus to be deleted are amplified by polymerase chain reaction (PCR) and seamlessly fused with the linearized pEX18Tc sacB suicide plasmid by Gibson assembly. The resulting knockout plasmid is transformed into P. aeruginosa by an electroporation method optimized in this study. The plasmid is then integrated into the chromosome by homologous recombination, and knockout mutants are identified via sacB mediated sucrose counter-selection. The current method was employed to generate clean gene knockouts of the heme assimilation system anti-σ factor, hasS, and the virulence regulator involving ECF system anti-σ and σ factors vreA and vreI, respectively. The process from plasmid construction to confirmation by DNA sequencing of the gene knockout was completed in one week. Furthermore, the utility of the method is highlighted in the construction of the vreA and vreI knockouts, where the start codon of vreA and the stop codon of vreI overlap. Utilizing Gibson assembly knockout mutants were constructed with single base-pair precision to generate the respective vreA and vreI knockouts while maintaining the start and stop codon of the respective genes. Overall, this method allows for the rapid construction of gene KOs in P. aeruginosa with base-pair precision.

References:

Zeling Xu, Ming Li, Yanran Li, Huiluo Cao, Hua Xiang, Aixin Yan. Native CRISPR-Cas mediated in situ genome editing reveals extensive resistance synergy in the clinical multidrug resistant Pseudomonas aeruginosa. bioRxiv 496711.

Weizhong Chen, Ya Zhang, Yifei Zhang, Yishuang Pi, Tongnian Gu, Liqiang Song, Yu Wang, Quan jiangJi. iScience, Volume 6, 31 August 2018, Pages 222-231.

Huang, Weiliang, and Angela Wilks. “A rapid seamless method for gene KO in Pseudomonas aeruginosa.” BMC microbiology vol. 17,1 199. 19 Sep. 2017, doi:10.1186/s12866-017-1112-5.