Solutions

Pseudomonas putida CRISPR-Cas9 Genome Editing

Precise CRISPR-Cas9 genome editing for Pseudomonas putida, from single-gene deletions to iterative strain engineering.

Custom guide RNA design
Single-plasmid system for rapid editing
Sequence-verified edited clones

Our service leverages a CRISPR-Cas9 system tailored for Pseudomonas putida, enabling efficient genome modifications from single-gene deletions to iterative multi-locus engineering. The workflow integrates guide RNA design, plasmid construction, transformation, colony screening, and sequence verification — all managed by our experienced team to deliver validated edited strains.

Gene deletion, insertion, and replacement
Verified by PCR and Sanger sequencing
Iterative editing through plasmid curing

Host-optimized Cas9

Single-plasmid delivery

Plasmid curing for iterative cycles

Our Approach

Streamlined Genome Editing for Pseudomonas putida

We combine custom gRNA design, single-plasmid CRISPR-Cas9 delivery, and robust assessment to deliver precisely edited strains for your research or production goals.

Streamlined Design-Build-Test Cycle

We design gRNAs and construct the all-in-one plasmid, then transform into P. putida for editing.

In silico gRNA design with off-target scoring
Single-plasmid CRISPR-Cas9 construct assembly
Transformation into P. putida with selection

Robust Editing Verification

Edits are confirmed by PCR and Sanger sequencing across the target locus.

Colony PCR screening for edit junctions
Sanger sequencing of target region
Plasmid curing assessment

Rapid Iterative Editing

The single-plasmid system allows easy curing between cycles, enabling sequential modifications in the same strain.

Precision Gene Deletions and Insertions

Both small deletions and targeted insertions (up to several kb) are achievable via homology-directed repair templates.

Comprehensive QC

Each edited clone is sequence-verified; off-target analysis is available as an optional add-on.

Deliverables

What You Receive

Standard deliverables include sequence-confirmed edited strains and project documentation. Additional options extend your analysis.

Standard Deliverables

Sequence-confirmed edited P. putida strain(s)
Edited clone documentation (PCR gel images, sequencing chromatograms)
CRISPR plasmid map and sequence file
Project summary report with gRNA design details

Optional Add-Ons

Additional clones for screening
Off-target analysis (in silico and targeted amplicon sequencing)
Expression characterization of edited genes
Library generation for large-scale editing

Service Scope

Parameter	Typical project scope
Edit Type	Gene deletion, insertion, and replacement (scoped per project after consultation)
Donor Template	Synthetic DNA with homology arms (length and design scoped per project)

Workflow

From Design to Validated Strain

Our five-step process ensures efficient and accurate genome editing in Pseudomonas putida.

Guide Design

We design specific sgRNAs targeting your gene of interest, with on-target efficiency and off-target potential evaluated computationally.

Construct Assembly

A single-plasmid expressing Cas9 and the sgRNA, along with a homology-directed repair template when needed, is assembled and sequence-verified.

Transformation

The plasmid is transformed into P. putida via electroporation; edited cells are selected using antibiotic or fluorescent markers.

Colony Screening

Individual colonies are screened by colony PCR to identify those carrying the desired edit at the target locus.

Verification and Curing

Editing is confirmed by Sanger sequencing, then the CRISPR plasmid is cured to enable a clean strain for subsequent rounds or applications.

Applications

Serving Diverse Research Goals

Our Pseudomonas putida genome editing service supports metabolic engineering, functional genomics, and biocontainment projects.

Pathway Optimization

Our service supports targeted knockout of competing pathways or insertion of biosynthetic genes.

Knockout of competing metabolic branches
Insertion of heterologous genes for novel products
Iterative editing to fine-tune flux

Gene Function Studies

Generate precise deletion mutants to investigate gene essentiality and phenotype.

Single-gene knockout libraries
Complementation studies with knock-in
Validation of predicted essential genes

Safeguard Systems

Design CRISPR-based kill switches or auxotrophic markers for safe industrial strains.

Essential gene targeting for conditional lethality
Repetitive element cleavage for genotoxic response
Iterative editing for multi-layer containment

Frequently Asked Questions

Turnaround is scoped per project based on edit complexity and number of clones required. We provide a timeline during the project consultation and confirm it in the SOW.
Editing is confirmed by colony PCR screening followed by Sanger sequencing of the target region. We sequence both junctions and the full edited locus to support correct modification. For insertions, we investigate presence and correct orientation of the inserted sequence. Plasmid curing is confirmed by loss of antibiotic resistance and further PCR.
Yes, the single-plasmid system is designed for rapid curing between rounds. After one edit is verified and the plasmid cured, a new CRISPR plasmid targeting the next locus can be introduced. This iterative approach allows sequential addition or removal of genes without leaving residual selection markers.
Turnaround depends on the complexity of the edit and the number of clones needed. For a standard gene deletion with one target clone, the timeline is scoped during project consultation. We aim to provide sequence-verified constructs with strain confirmation scoped per project a timeline confirmed during project scoping, but exact timelines are confirmed in the project SOW based on your specific requirements.

Scientific Literature

References

Wen Q, Chen J, Li J, et al. A single-plasmid-based, easily curable CRISPR/Cas9 system for rapid, iterative genome editing in Pseudomonas putida KT2440. Microbial cell factories. 2024;23(1):349. View on PubMed
Asin-Garcia E, Martin-Pascual M, de Buck C, et al. GenoMine: a CRISPR-Cas9-based kill switch for biocontainment of Pseudomonas putida. Frontiers in bioengineering and biotechnology. 2024;12:1426107. View on PubMed
Liu S, Narancic T, Davis C, et al. CRISPR-Cas9 Editing of the Synthesis of Biodegradable Polyesters Polyhydroxyalkanaotes (PHA) in Pseudomonas putida KT2440. Methods in molecular biology (Clifton, N.J.). 2022;2397:341-358. View on PubMed
Sharaev N, Chacon-Machado L, Musharova O, et al. [Repair of Double-Stranded DNA Breaks Generated by CRISPR-Cas9 in Pseudomonas putida KT2440]. Molekuliarnaia biologiia. 2022;56(6):914. View on PubMed
Wirth NT, Kozaeva E, Nikel PI. Accelerated genome engineering of Pseudomonas putida by I-SceI-mediated recombination and CRISPR-Cas9 counterselection. Microbial biotechnology. 2020;13(1):233-249. View on PubMed

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