Klebsiella pneumoniae Phage Genome Editing Service

Klebsiella pneumoniae Phage Genome Editing Service

1. Research Background

Klebsiella pneumoniae is a clinically common Gram-negative opportunistic pathogen, and the widespread spread of carbapenem-resistant Klebsiella pneumoniae (CRKP) in particular has become a major global public health threat. As viruses that can precisely kill specific pathogenic bacteria, phages hold enormous potential in the treatment of drug-resistant bacterial infections. However, natural phages are often limited by narrow host ranges and weak biofilm penetration. Precise and efficient genome editing technologies serve as core tools for modifying phage properties and expanding their applications in clinical treatment and detection.

Against this backdrop, GeneRulor launches customized modification services for Klebsiella pneumoniae phages. Utilizing the CRISPR/Cas9 negative selection system or BRED (Bacteriophage Recombineering of Electroporated DNA) technology, we can achieve precise target gene knockout, site-directed mutagenesis, and exogenous gene integration (e.g., capsular depolymerase integration) in the phage genome, and deliver positive mutant phage strains purified through multiple rounds.

2. Host Characteristics and Biological Background

（1）Host Cell Wall and Capsule Properties: The most prominent feature of Klebsiella pneumoniae is its thick polysaccharide capsule. This barrier not only protects the bacterium from immune system attacks but also determines the specificity of phage recognition.

（2）Clinical Context: Research on CRKP and hypervirulent Klebsiella pneumoniae (hvKp) is currently the core of clinical challenges. Edited phages can specifically disrupt the capsules of specific serotypes.

（3）Phage Diversity: Including Siphoviridae (long-tailed), Podoviridae (short-tailed) and Myoviridae (contractile-tailed) phages, which often carry a variety of depolymerases to penetrate bacterial capsules.

Figure 1 Infection of host cells by Klebsiella pneumoniae phages

3. Reported Editing Strategies

In light of the characteristics of rapid replication and lysis of the Klebsiella pneumoniae phage genome in host cells, the current mainstream editing strategies include:

3.1 CRISPR/Cas9-assisted selection system (core protocol):

（1）Principle: Cas9 and sgRNAs targeting wild-type phages are pre-introduced into host bacteria. When phages infect the host, Cas9 cleaves the wild-type genome, while edited strains that have undergone homologous recombination survive due to altered target sites.

（2）Advantages: It greatly improves the extremely low mutation rate of traditional homologous recombination (usually below 10⁻⁴), enabling highly efficient screening.

3.2 Homologous Recombination:

The host bacterial recombination system (e.g., the λ-Red system) is used to introduce donor DNA containing mutant sequences during phage DNA replication.

3.3 In Vitro Assembly and Rebooting:

The phage genome is fragmented for in vitro modification and ligation, then introduced into competent host cells via electroporation for viral particle packaging.

4. Core Application Fields

（1）Host Range Expansion: Alter the host recognition specificity of phages through site-directed mutagenesis or domain swapping of tail fiber proteins or receptor-binding proteins (RBPs).

（2）Enhanced Biofilm Degradation: Integrate potent capsular depolymerases or depolymerase gene clusters into the phage genome to enhance its ability to destroy biofilms of clinical drug-resistant strains.

（3）Synthetic Biology Modification: Integrate fluorescent protein or luciferase genes for the rapid detection of Klebsiella pneumoniae in clinical samples.

（4）Functional Genomics: Precisely delete non-essential phage genes or lysis-related genes to investigate their infection mechanisms.

5. Project Process and Validation

We offer one-stop services from design to delivery to ensure the accuracy of editing results:

（1）Protocol design and vector construction: Design mutation templates, sgRNAs targeting wild-type phages, and corresponding screening plasmids.

（2）Host Bacterium Engineering: Introduce the CRISPR system and donor DNA into recipient Klebsiella pneumoniae strains.

（3）Phage Screening and Enrichment: Wild-type phages infect engineered bacteria, and edited progeny phages are enriched using Cas9 selection pressure.

（4）Single Plaque Validation: Perform 3–5 consecutive rounds of single plaque purification, combined with PCR identification and whole-genome sequencing to confirm positive mutations.

Figure 2 Schematic of the Project Process

6. Introduction to Genome Editing Projects

6.1 Our core services include:

（1）Gene Knockout/Inactivation: Precisely delete unwanted virulence-related genes or non-essential fragments.

（2）Gene Integration/Overexpression: Insert exogenous sequences such as depolymerases and fluorescent probes into intergenic regions or non-essential gene loci of the genome.

（3）Precise Site-Directed Mutagenesis: Perform site-specific modification of key amino acids in RBP proteins.

（4）Multi-Locus Editing: Realize sequential modification of multiple tail proteins or regulatory genes.

6.2 Technical Advantages

（1）High Specificity: Rich experience in handling a variety of host bacterial serotypes (K1, K2, K47, K64, etc.) and their corresponding phages.

（2）High-Efficiency Screening: The optimized CRISPR selection system can increase the screening efficiency to over 80%.

（3）Fast Delivery Cycle: Rely on standardized processes to ensure the delivery of verified mutant phages in the shortest possible time.

7. Case Introduction

Case: Antimicrobial Potential of a Novel K5-Specific Phage and Its Recombinant Strains Against Klebsiella pneumoniae in Milk

Project Content: Modified and systematically evaluated the antimicrobial effects of a novel K5-specific phage and its engineered recombinant strains against Klebsiella pneumoniae in a milk environment.

（1） Research Background

Milk is rich in nutrients, providing an ideal environment for bacterial proliferation, among which Klebsiella pneumoniae is an important pathogen causing bovine mastitis and dairy product contamination. With the increasingly serious problem of antibiotic resistance (e.g., the emergence of carbapenem and colistin-resistant strains), phage therapy has become an alternative strategy. However, most existing studies focus on K1 and K2 serotypes; phage resources targeting the hypervirulent K5 serotype, which is prevalent in dairy farms in China, are scarce, and their application potential in dairy products has not been fully explored. This study aimed to fill this gap and provide new ideas for dairy product safety by developing K5-specific phage tools.

（2）Protocol Design

The research team constructed and optimized phage resources through a multi-step engineering strategy:

Phage Isolation and Identification: K1-specific phages P284, P287 and K5-specific phage P252 were isolated from hospital sewage, and their morphologies were confirmed by electron microscopy (e.g., P252 is a short-tailed phage with a head diameter of 58±0.5 nm). Genome sequencing showed that these phages contain no lysogenic, virulence or resistance genes, complying with biosafety requirements.

Receptor-Binding Protein (RBP) Engineering: The CRISPR-Cas9 system was used for targeted editing of the phage genome, replacing the RBP of K1-specific phage P284 with that of K5-specific phage P252, and successfully generating recombinant phages T and F. This method realized the host range switch from K1 to K5 through homologous recombination, significantly expanding the diversity of the phage library.

Activity Evaluation System: The antimicrobial effects of phages at different multiplicities of infection (MOI=10, 1, 0.1) were systematically evaluated through planktonic culture assays and milk models (4°C simulating refrigeration, 38°C simulating room temperature).

（3）Experimental Conclusions

Recombinant Phages Exhibit Superior Antimicrobial Activity: In planktonic culture assays, recombinant phages T and F showed significantly better inhibitory effects on K5 strains than the wild-type P252 at MOI=10, reducing bacterial counts by up to 2.99 log₁₀ cfu/mL (P<0.001), and the inhibitory effect was more durable (remaining highly effective within 12 hours). Recombinant phages could still form degradation halos against some non-lytic K5 strains, indicating that their RBPs can specifically recognize capsular polysaccharides.

Efficient Bacterial Inhibition in Milk Environment: Under 4°C refrigeration conditions, all K5-specific phages (P252, T, F) significantly reduced bacterial load within 9 hours (up to a 2.70 log₁₀ cfu/mL reduction), and the inhibitory effect was positively correlated with MOI. At 38°C, the phage-treated groups completely inhibited bacterial fermentation (severe fermentation occurred in the control group), proving their application potential at different temperatures.

Safety Verification: Genomic analysis confirmed no insertion of harmful genes in recombinant phages, and their specific lysis spectrum avoided impacts on non-target microbial communities.

（4） Application Significance

Contamination Control in the Dairy Industry: This study is the first to demonstrate the effectiveness of K5-specific phages under refrigerated and room temperature conditions in milk, providing a novel biological control tool for preventing dairy product spoilage and bovine mastitis. The engineering strategy of recombinant phages can be extended to other serotypes to achieve precise prevention and control with a "one strain, one strategy" approach.

Expansion of Phage Therapy Resources: CRISPR-Cas9-mediated host range modification provides a technical paradigm for the rapid construction of targeted phage libraries, overcoming the approval complexity of traditional phage cocktail therapy.

Future Directions: Further evaluation of phage stability in real dairy product production chains is needed, and the potential for combination use with depolymerases should be explored to enhance the clearance efficiency of drug-resistant strains.

8. References

[1] Bari, S. M., et al. (2017). Orthogonal Cas9 proteins for RNA-guided gene regulation and editing. Nature Methods.

[2] Sun, Y., et al. (2022). Engineering Klebsiella pneumoniae phages to enhance their bactericidal and biofilm-removal activity. Frontiers in Microbiology.

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

[4] Luo, M. L., et al. (2016). The CRISPR-Cas9 system as a tool for editing the genomes of Staphylococcus aureus. Applied and Environmental Microbiology.

[5] Li, P., et al. (2025). Antimicrobial potential of a novel K5-specific phage and its recombinant strains against Klebsiella pneumoniae in milk. Journal of Dairy Science.

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