CD Biosynsis provides high-precision Genome Editing Services for Pseudomonas putida using the powerful CRISPR-Cas9 system. P. putida is a premier chassis for biocatalysis and metabolic engineering, but its complex genome necessitates robust and specific editing tools. Our service leverages optimized CRISPR-Cas9 delivery and repair mechanisms (such as homologous recombination) to perform highly efficient gene knockouts, precise gene knock-ins, single nucleotide polymorphisms (SNPs), and promoter tuning. This technology drastically accelerates the Design-Build-Test-Learn (DBTL) cycle, enabling rapid development of high-performance P. putida strains for chemical synthesis and environmental applications.
Get a QuoteP. putida is known for its resilience and diverse metabolic capacity, making it a highly desirable host in synthetic biology. However, conventional genetic tools often involve lengthy multi-step cloning and low-efficiency screening. Our CRISPR-Cas9 platform overcomes these limitations by utilizing targeted double-strand breaks (DSBs) guided by single guide RNA (sgRNA). This approach ensures unparalleled editing specificity and efficiency, particularly when combined with optimized homologous recombination strategies. The result is rapid, multiplex, and scarless modification of the P. putida genome, which is crucial for complex metabolic pathway engineering where dozens of genetic changes are required.
Scarless Deletions
Complete, marker-free removal of target genes using a donor template, eliminating metabolic burden and resistance genes.
Multiplex Editing
Simultaneous deletion of multiple genes (e.g., competing pathways) in one step, accelerated optimization of complex pathways.
Non-Essential Gene Targeting
Targeting genes for side-product formation, protease excretion, or improved substrate utilization.
Pathway Integration
Precise, stable integration of large biosynthetic gene clusters or heterologous pathways into safe harbor loci.
Point Mutations (SNPs)
Introducing single nucleotide changes to modify enzyme activity or change protein functionality.
Tagging and Reporting
Insertion of fluorescent tags (e.g., GFP) or affinity tags for protein visualization and purification.
Promoter Replacement
Replacing native promoters with synthetic promoters of defined strength (strong, medium, weak) to optimize flux through a pathway.
RBS Optimization
Engineering the Ribosome Binding Site (RBS) to fine-tune translational efficiency and protein levels.
Our validated process ensures high-throughput design, efficient transformation, and precise validation of all edits.
1. Design & Synthesis
2. Delivery & Editing
3. Screening & Selection
4. Validation & Curing
Bioinformatic analysis for target identification (gene, promoter, SNP).
Design and synthesis of high-specificity sgRNA and homologous recombination donor templates.
Cloning of editing components into optimized P. putida delivery vectors.
Efficient introduction of the CRISPR system (Cas9, sgRNA, donor) into the target strain, typically via conjugation or electroporation.
Induction of Cas9 expression to initiate targeted double-strand break (DSB).
Genotype Verification: Full Sanger sequencing of the edited genomic locus to confirm precise, scarless modification.
Plasmid Curing: Removing the Cas9/sgRNA plasmid to ensure the final strain is stable and editing is permanent.
Deliver the verified, marker-free engineered strain and comprehensive data report.
High Efficiency in P. putida
Use of optimized Cas9 variants and delivery methods specifically tuned for the P. putida genetic background.
Multiplex Engineering
Capability to simultaneously edit up to five targets, which is critical for redesigning large metabolic pathways quickly.
Scarless and Marker-Free
Final strains are free of any foreign DNA, ensuring regulatory compliance and maximizing strain stability and fitness.
Rapid DBTL Acceleration
The precision and speed of CRISPR significantly reduce the time needed for strain construction, accelerating R&D cycles.
How does CRISPR-Cas9 achieve scarless editing?
CRISPR-Cas9 creates a precise break in the DNA. We supply a donor DNA template without resistance markers. The cell repairs the break using this template via Homologous Recombination, resulting in the desired, marker-free edit.
Is the final engineered strain plasmid-free?
Yes. The Cas9 and sgRNA plasmids are designed to be temporary. After successful genome integration, a counter-selection step is performed to "cure" the strain, removing the editing tools and ensuring the final strain is stable and contains only the intended genomic modification.
What is the typical turnaround time for a single edit?
A typical single, validated CRISPR-Cas9 edit (from design to final strain delivery) usually takes between 4 to 6 weeks, depending on the complexity of the target locus and the specific P. putida strain background.
Can this service be used on different P. putida strains (e.g., KT2440)?
Yes. Our platform is built on established editing protocols for common laboratory strains like P. putida KT2440 and can be adapted to other specialized wild-type or engineered derivatives.
CRISPR-Cas9 technology represents a transformative advancement in gene editing techniques. The main function of the system is to precisely cut DNA sequences by combining guide RNA (gRNA) with the Cas9 protein. This technology became a mainstream genome editing tool quickly after its 2012 introduction because of its efficient, simple and low-cost nature.
The CRISPR gene editing system with its Cas9 version stands as a vital instrument for current biological research. CRISPR technology enables gene knockout (KO) through permanent gene expression blockage achieved by sequence disruption. Various scientific domains including disease modeling and drug screening employ this technology to study gene functions. CRISPR KO technology demonstrates high efficiency and precision but requires confirmation and verification post-implementation because unsatisfactory editing may produce off-target effects or incomplete gene knockouts which impact experimental result reliability. For precise and efficient Gene Editing Services - CD Biosynsis, Biosynsis offers comprehensive solutions tailored to your research needs.
The CRISPR-Cas9 knockout cell line was developed using CRISPR/Cas9 gene editing to allow scientists to remove genes accurately for research on gene function and disease models and pharmaceutical discovery. Genetic research considers this technology essential due to its high efficiency together with simple operation and broad usability.
If your question is not addressed through these resources, you can fill out the online form below and we will answer your question as soon as possible.
CD Biosynsis is a leading customer-focused biotechnology company dedicated to providing high-quality products, comprehensive service packages, and tailored solutions to support and facilitate the applications of synthetic biology in a wide range of areas.