K. pneumoniae Engineered Phage Development & Genome Editing

OverviewServicesSample RequirementsAdvantagesApplicationsCase StudyFAQs

Overview

Service Overview

Klebsiella pneumoniae is a clinically prevalent Gram-negative opportunistic pathogen. The global spread of carbapenem-resistant K. pneumoniae (CRKP) and hypervirulent lineages (hvKp) has made it a leading public-health threat, with limited therapeutic options and rising mortality across hospital and community settings.

Natural phages offer precision killing of drug-resistant bacteria, but their narrow host range, weak biofilm penetration, and capsule-determined specificity constrain clinical use. Gene editing is the core tool to broaden host range, integrate depolymerases, and expand diagnostic and therapeutic applications against this pathogen.

Creative BioMart Microbe applies CRISPR-Cas9 counter-selection, λ-Red homologous recombination, and in vitro assembly to edit K. pneumoniae phages with high efficiency. From capsule-serotype retargeting to multi-locus engineering, our workflow delivers validated mutant phage and hands off to phage fermentation and large-scale production. Explore our broader Phage Genome Editing Services for the full platform overview.

Platform overview of Klebsiella pneumoniae phage genome editing: the capsular barrier as the central engineering target, three complementary editing platforms (CRISPR-Cas9 counter-selection, lambda-Red recombineering, in vitro assembly), application endpoints, and deliverables.
Figure 1. K. pneumoniae phage editing platform overview — the capsular barrier as the central engineering target, three complementary editing platforms, and the application and deliverable endpoints they enable.

Host Characteristics & Biological Background

Engineering K. pneumoniae phages must contend with the capsule — the defining barrier that shapes both virulence and phage recognition. The table below frames the three features that drive our editing strategy.

Feature	Detail	Editing Implication
Polysaccharide capsule	The thick capsule is the defining surface barrier and the primary determinant of phage receptor recognition.	Engineering tail-fiber or receptor-binding proteins, or integrating depolymerases, overcomes serotype-specific restriction.
Clinical context	CRKP and hypervirulent K. pneumoniae (hvKp) are the current research frontier.	Edited phages target capsule destruction of specific serotypes for therapeutic and assay-development research.
Phage diversity	Phages of siphovirus-like (long non-contractile tail), podovirus-like (short tail), and myovirus-like (contractile tail) morphotypes, often carrying multiple depolymerases.	Strategy is chosen per morphology and depolymerase repertoire to maximize host coverage.

Services

Service Workflow

Each project follows a six-step pipeline — from project consultation to a validated, sequence-confirmed mutant phage — with the editing strategy customized to your phage and host.

Horizontal six-step workflow banner for Klebsiella pneumoniae phage editing: project consultation, strategy design, editing-host preparation (Cas9/sgRNA), genome editing and counter-selection enrichment, single-plaque purification, sequencing validation and delivery.

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Editing Strategies

Strategy	Mechanism	Used For
CRISPR-Cas9 counter-selection	Cas9 and sgRNA targeting the wild-type genome are pre-introduced into the host; edited recombinants survive cleavage because their target sites are altered.	Point mutations, knockouts, and insertions against the traditionally low homologous-recombination rate (below 10⁻⁴).
λ-Red recombineering (homologous recombination)	The host recombination system (e.g., λ-Red) introduces mutagenic donor DNA during phage genome replication.	In-frame deletions, small insertions, and cargo integration into intergenic loci.
In vitro assembly & rebooting	The genome is fragmented, modified, and ligated in vitro, then electroporated into competent host cells for virion packaging.	Large modular swaps, multi-locus constructs, and whole-genome recoding.

Service Details

3D illustration of a Klebsiella pneumoniae phage with a deleted gene region and a sequencing readout confirming the edit.

Gene Knockout / Inactivation

We delete non-essential, lysogeny-associated, or otherwise unwanted K. pneumoniae phage genes to dissect gene function or streamline the genome. Using CRISPR-Cas9 counter-selection, edited genomes survive cleavage of the wild-type target while recombinants are enriched. Each knockout is confirmed by PCR and whole-genome sequencing, delivered as a plaque-purified lysate with a full editing report.

3D illustration of a Klebsiella pneumoniae phage with an inserted depolymerase enzyme module glowing within its tail structure.

Gene Integration / Overexpression

We insert exogenous sequences, such as capsule depolymerases or fluorescent reporters, into intergenic or non-essential loci of the K. pneumoniae phage genome. Donor templates are introduced during infection, and Cas9 selection suppresses the wild-type background. Integrated cargo is verified by sequencing and functional assay, supporting biofilm degradation or rapid clinical detection.

3D illustration of a Klebsiella pneumoniae phage receptor-binding protein with a highlighted amino-acid mutation site on its tail fiber.

Precision Point Mutation

We introduce site-directed amino-acid changes in receptor-binding proteins to alter host recognition or enzymatic activity. A mutagenic oligo and sgRNA are co-delivered; mutants with altered target sites evade Cas9 cleavage and are enriched. Each point mutant is plaque-purified and Sanger- or whole-genome-sequenced to confirm the precise substitution.

3D illustration of a Klebsiella pneumoniae phage with multiple edited tail-protein and regulatory modules shown as labeled nodes along its genome.

Multi-Locus Editing

We perform sequential or synchronous modification of multiple tail proteins, regulatory genes, or depolymerase clusters across the K. pneumoniae phage genome. Each edit layer is selected and verified before the next, preserving viability. The result is a multi-engineered phage with expanded host range and enhanced biofilm activity, delivered with per-locus sequencing validation.

Deliverables & QC

Plaque-purified, high-titer edited K. pneumoniae phage lysate.
PCR identification and whole-genome or Sanger sequencing confirmation of the edit.
Wild-type background and off-target assessment by sequencing.
Host-range and capsule-serotype characterization across supplied isolates.
Project report with sgRNA or primer sequences, donor-template maps, and methods.

Sample Requirements

To initiate a project, provide as much of the following as possible. Our team will advise on any gaps during the consultation.

Required Information	Optional Information	Not Accepted
Target phage (isolated lysate or deposited strain) and/or host K. pneumoniae isolate Desired edit: gene knockout, point mutation, integration, or host-range change, with target coordinates where known Intended application and downstream use (research, therapeutic, industrial)	Existing genome sequence or annotation of the phage or host Preferred editing strategy or prior-attempt data Screening or bactericidal assay protocols you want mirrored	Pathogenic strains restricted by local containment law without prior approval Projects requiring BSL-3 containment (determined by the host strain) unless pre-authorized Samples lacking any host or sequence context

Recommended Input by Edit Type

Edit Type	Preferred Input	Typical Timeline
Point mutation / RBP modification	Phage lysate + K. pneumoniae host + target gene or protein	3–5 weeks
Depolymerase / reporter integration	Phage + donor sequence + intergenic target	4–6 weeks
Multi-locus editing	Phage + host + list of loci to modify	6–10 weeks
Host-range / serotype expansion	Phage + multiple K. pneumoniae isolates	4–8 weeks

Timelines are typical estimates for standard projects and are confirmed per project after consultation; actual duration may vary with phage and host characteristics.

Lysates should be shipped on dry ice with glycerol backups; genomic DNA is accepted in ethanol or TE at 4°C. Contact our team via contact us before sending BSL-2 materials.

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Our Advantages

Broad serotype applicability — Our editing platforms apply to phages targeting the major clinical capsular serotypes of K. pneumoniae (including K1, K2, K47, and K64), supporting engineering against diverse clinical isolates.
High-efficiency screening — Optimized CRISPR-Cas9 counter-selection converts the traditionally low homologous-recombination rate (below 10⁻⁴) into efficient recovery of edited mutants, with the large majority of screened plaques carrying the intended edit.
Fast delivery cycle — A standardized workflow delivers confirmed mutant phage within the shortest feasible timeframe, with milestones from design to validated clone tracked transparently.
CRKP & hvKp clinical-isolate expertise — Deep focus on carbapenem-resistant and hypervirulent K. pneumoniae, including capsule-targeted engineering for the strains driving current clinical outbreaks.
GMP-compatible scale-up — Edited phages hand off to phage fermentation and large-scale production under documentation-ready workflows for therapeutic translation.

Applications

3D icon of a Klebsiella pneumoniae phage tail receptor binding to multiple capsule-serotype bacterial cells representing host-range expansion.

Host-Range Expansion

Tail-fiber and receptor-binding protein point mutations or domain swapping alter K. pneumoniae recognition, letting a single engineered phage cover multiple capsular serotypes and clinical isolates.

3D icon of a Klebsiella pneumoniae biofilm being broken open by a phage-encoded depolymerase enzyme representing biofilm degradation.

Enhanced Biofilm Degradation

Integrating potent capsule depolymerases into the phage genome strengthens destruction of mature biofilms formed by drug-resistant K. pneumoniae, improving penetration and killing.

3D icon of a Klebsiella pneumoniae phage carrying a fluorescent reporter gene for a rapid bacterial detection assay.

Synthetic Biology Engineering

Fluorescent proteins or luciferase genes inserted into phage genomes enable rapid, phage-based detection of K. pneumoniae, supporting research use and diagnostic-assay development.

3D icon of a Klebsiella pneumoniae phage genome with highlighted deleted non-essential genes representing functional genomics.

Functional Genomics

Precise deletion of non-essential or lysis-related phage genes dissects infection, receptor recognition, and progeny-packaging mechanisms in K. pneumoniae systems.

Case Study

Case Study 1: Modular Receptor-Binding Protein Engineering Switches Klebsiella Phage Capsule Serotype Specificity

Klebsiella phages recognize their hosts through receptor-binding proteins (RBPs) built from an N-terminal structural module anchoring the protein to the tail and a C-terminal specificity module with depolymerase activity against a defined capsular serotype. To mimic the natural horizontal transfer of these modules, chimeric RBPs exchanging anchor and specificity modules were assembled and transplanted into a phage K11 scaffold. All chimeras strictly followed the capsular serotype specificity of the C-terminal module: synthetic phages carrying KP32- or KP34-derived specificity modules switched from the K11 serotype to K3, K21, or K63, with a corresponding host-range change, while anchor swaps alone preserved the original specificity. The work establishes modular RBP exchange as a practical route to retarget Klebsiella phages to new capsular serotypes.

Figure 2. Specificity of the constructed synthetic phages against Klebsiella strains with different capsular serotypes. (Latka, et al. 2021)

FAQs

Q: Why is the K. pneumoniae capsule such a barrier to phage editing and therapy?

A: The thick polysaccharide capsule is both a virulence factor and the primary determinant of phage receptor recognition. It physically blocks adsorption and shields the cell from immune attack, so most phages are restricted to a single capsular serotype. Our editing targets tail-fiber and receptor-binding proteins or integrates depolymerases to overcome this serotype-specific barrier.

Q: How does CRISPR-Cas9 counter-selection improve editing efficiency in K. pneumoniae phages?

A: Traditional homologous recombination in phage genomes is extremely rare, often below 10⁻⁴. By pre-introducing Cas9 and a wild-type-targeting sgRNA into the host, we cleave unmutated phage genomes during infection; only recombinants with altered target sites survive. This negative selection greatly enriches edited mutants among recovered clones.

Q: Can you engineer phages to cover multiple capsular serotypes such as K1, K2, K47, and K64?

A: Yes. Our editing platforms apply to phages targeting the major clinical capsular serotypes, including K1, K2, K47, and K64, and we use receptor-binding-protein domain swapping or multi-locus editing to broaden a single phage's host range. Each engineered derivative is characterized against the specific clinical isolates you supply.

Q: How do you handle carbapenem-resistant (CRKP) and hypervirulent (hvKp) strains safely?

A: Edited phages are developed under BSL-2 containment appropriate to the host, with strains documented and permitted under local rules. Each edited phage genome is verified by whole-genome sequencing to exclude unintended cargo, virulence-factor, or mobilizable elements. BSL-3-restricted hosts require prior authorization before project initiation.

Q: What deliverables and validation do edited K. pneumoniae phages receive?

A: Every edited phage is taken through 3–5 rounds of single-plaque purification, then confirmed by PCR identification and whole-genome sequencing. Deliverables include a plaque-purified lysate, wild-type background and off-target assessment, host-range characterization, and a project report with sgRNA, primer, and donor-template maps.

Q: Do you support multi-locus or multi-gene editing in one project?

A: Yes. We perform sequential or synchronous modification of multiple tail proteins, regulatory genes, or depolymerase clusters across the phage genome. Each edit layer is selected and verified before the next to preserve viability, and the final multi-engineered phage ships with per-locus sequencing validation.

Q: How do edited K. pneumoniae phages transition to larger-scale production?

A: Validated edited phages hand off to our phage fermentation and large-scale production service, which operates documentation-ready, GMP-compatible workflows for therapeutic and industrial scale-up of clinical K. pneumoniae phage candidates.

References:

Latka, A., et al. (2021). Engineering the modular receptor-binding proteins of Klebsiella phages switches their capsular serotype specificity. mBio, 12(3), e00455-21.

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