Pseudomonas aeruginosa Genome Editing Service

Pseudomonas aeruginosa Genome Editing Service

Pseudomonas aeruginosa is an extremely important Gram-negative opportunistic pathogen in clinical settings, renowned for its robust metabolic capacity, high-level antibiotic resistance (e.g., MDR and PDR strains), and sophisticated quorum sensing system. Targeting its complex genome and highly efficient repair mechanisms, GeneRulor leverages advanced CRISPR/Cas9 and homologous recombination technologies to provide precise genome modification services, including gene knockout, site-directed mutagenesis and fragment integration, facilitating in-depth research on drug resistance mechanisms and pathogenicity.

1. Bacterial Characteristics and Biological Background

Gram Staining Property: Pseudomonas aeruginosa is a Gram-negative bacterium. Unlike Staphylococcus aureus, it has a thin peptidoglycan layer in the cell wall, but is equipped with a complex outer membrane structure and efflux pump system.

Clinical Significance: It is one of the major pathogens responsible for nosocomial infections, often causing respiratory tract infections (especially in cystic fibrosis patients), burn wound infections and sepsis. Its extensive drug resistance spectrum makes the development of novel drug targets an urgent priority.

Metabolism and Applications: With strong environmental adaptability, it serves as a standard model organism for studying quorum sensing, biofilm formation and secretion systems (e.g., T3SS, T6SS).

2. Reported Editing Strategies

Targeting the highly efficient restriction-modification system and complex cell envelope of Pseudomonas aeruginosa, we adopt the following mainstream protocols:

CRISPR/Cas9 or Cas12a System:

Principle: Cas proteins are guided by sgRNAs to precisely cleave genomic DNA, inducing double-strand breaks (DSB).

Advantages: Combined with donor DNA, it enables scarless editing, highly efficient gene knockout or large-fragment integration.

Double Homologous Recombination (Double Crossover Method):

Suicide plasmids carrying negative selection markers such as sacB are used to achieve precise substitution or deletion of target bases through two-step crossover.

Transformation Optimization with Auxiliary Plasmids:

Aiming at the low transformation efficiency of clinical drug-resistant strains, electroporation procedures are optimized or conjugation is adopted to overcome physical barriers.

Figure 1. Efficient C-to-T Conversion in Pseudomonas aeruginosa via the CRISPR/Cas9n-BEC Base Editor^[5]

3. Core Application Fields

Functional Genomics: Construct mutant libraries in large quantities to screen core genes associated with pathogenicity or metabolism.

Drug Resistance Mechanism Research: Simulate mutations in clinically isolated strains (e.g., gyrA, mexR mutations) through site-directed mutagenesis to elucidate the mechanisms of antibiotic resistance.

Synthetic Biology Modification: Insert fluorescent reporter genes into specific chromosomal loci (e.g., attB site) to track the colonization dynamics of bacteria in hosts in real time.

Engineered Bacterium Construction: Modify attenuated strains as vaccine vectors or optimize the synthesis of secondary metabolites in industrial strains.

4. Project Process and Validation

We offer full-process services from strategy design to mutant strain delivery:

Protocol Design: Design optimal target sites and homologous arms for PAO1, PA14 or clinically isolated strains.

Vector Construction: Synthesize sgRNAs and donor DNA, and construct high-efficiency editing plasmids.

Bacterial Transformation and Screening: Introduce plasmids via electroporation or conjugation, and screen positive clones through dual selection of antibiotic resistance and fluorescence.

Multiplex Validation: Conduct preliminary screening via colony PCR identification, and finally confirm the complete sequence correctness of the edited region through Sanger sequencing or whole-genome sequencing.

5. Introduction to Genome Editing Projects

Our core services include:

Gene Knockout/Inactivation: Precisely delete virulence factor or metabolic enzyme genes.

Gene Knock-in/Overexpression: Perform scarless insertion of exogenous promoters or reporter genes into the genome.

Precise Site-Directed Mutagenesis: Simulate single-base changes associated with clinical drug resistance.

Continuous Multi-Gene Editing: Realize simultaneous or sequential modification of multiple genes in complex pathways.

Technical Advantages

High Success Rate: Possess a mature operating system for both standard strains and clinical drug-resistant strains.

Scarless Editing: The finally delivered strains can be free of resistance markers and plasmid backbones, eliminating interference in subsequent experiments.

Full-Process Validation: Authentic and reliable data, with complete reports of electrophoresis images and sequencing peak maps provided.

Figure 2. Schematic Diagram of Project Workflow

6. Case Introduction

We have successfully provided services for numerous top universities, research institutions and biotech companies at home and abroad. A representative case is presented below:

Case: Successful Knockout of the Core Quorum Sensing Regulatory Gene lasR in Pseudomonas aeruginosa Standard Strain PAO1

Figure 3. Sequencing confirmation of successful target gene knockout

7. References

[1] Gorter, A. P., et al. (2021). Pseudomonas aeruginosa infections: Biology, resistance and therapy. Nature Reviews Microbiology.

[2] Hoang, T. T., et al. (1998). A broad-host-range flp-frt recombination system for site-specific excision of chromosomally-integrated DNA cassettes: applications for Pseudomonas aeruginosa. Gene.

[3] Chen, W., et al. (2017). CRISPR/Cas9-based genome editing in Pseudomonas aeruginosa and its application in studying drug resistance. Applied Microbiology and Biotechnology.

[4] Jiang, W., et al. (2013). RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nature Biotechnology.

Cooperate with Us

By choosing us, you will gain an experienced and technically proficient partner in genome editing. We commit to accelerating your research or projects with professional technologies, rigorous processes， and efficient communication.

Consult us now to obtain your customized editing scheme and quotation!