CD Biosynsis offers high-precision Targeted Gene Knock-in Services for Vibrio natriegens, a critical capability for converting this hyper-fast-growing organism into a reliable microbial cell factory. Gene knock-in involves the stable and precise integration of heterologous DNA—from single genes to entire biosynthetic pathways—into the host genome. Unlike unstable plasmid-based expression, genomic integration ensures long-term genetic stability and sustained expression across the host’s dual chromosomes. Utilizing advanced CRISPR-Cas9 mediated homologous recombination, our service specializes in high-efficiency, scarless integration into safe harbor loci. This accelerates the development of high-yield V. natriegens strains for synthesizing high-value chemicals, taking full advantage of the host's rapid growth rate for faster strain validation and scale-up.
Get a QuoteHarnessing the speed of V. natriegens for bioproduction requires not only rapid growth but also highly stable, efficient expression of the synthetic pathway. Plasmid-based systems are often prone to instability and metabolic burden, especially under non-selective industrial conditions. Our Gene Knock-in Service overcomes these limitations by utilizing precision CRISPR-Cas9 to guide the stable integration of desired DNA fragments into the host's genome. This ensures that the engineered pathway is permanent, consistently expressed, and balanced within the host's native, high-flux metabolism. We specialize in identifying and utilizing neutral safe harbor loci on both Chromosome I and Chromosome II to maximize genetic stability and achieve optimized gene dosage without impairing host fitness.
Plasmid-Free
Integration removes the need for antibiotic selection during large-scale fermentation and minimizes the metabolic burden associated with replicating high-copy plasmids.
Permanent Trait
The engineered phenotype is permanent and reliably inherited by all daughter cells, essential for robust industrial operation.
Large Fragment Knock-in
Capability to stably integrate large DNA fragments (up to 15 kb or more) containing entire synthetic operons or biosynthetic gene clusters (e.g., for polyketides or complex terpenoids).
Safe Harbor Targeting
Targeting neutral genomic sites to prevent unintended disruption of essential native host functions and ensure predictable expression.
Dual Chromosome Integration
Integration into specific loci on both Chromosome I and Chromosome II to achieve the desired gene copy number and expression level for pathway balancing .
We provide scarless, high-efficiency integration for diverse functional elements across the V. natriegens genome.
1. Heterologous Pathway Integration
2. Promoter & RBS Tuning
3. Reporter Gene Insertion
4. Dual Chromosome Targeting
Stable genomic knock-in of entire non-native biosynthetic pathways or large operons, ensuring uniform and high-level expression across the cell population.
Precise integration of synthetic promoters (inducible or constitutive) and optimized Ribosome Binding Sites (RBS) upstream of the target gene to fine-tune translational efficiency.
Insertion of fluorescent reporter genes (e.g., GFP, mCherry) or affinity tags (e.g., His-tag, Strep-tag) at specific loci for real-time monitoring and downstream protein purification.
Specialized protocols to target and integrate sequences into predetermined locations on both Chromosome I and Chromosome II to manage gene dosage and co-location.
1. Design & Donor Synthesis
Bioinformatic selection of the integration site and design of Cas9 sgRNA. Synthesis of the donor DNA template (gene/pathway + homology arms) for scarless integration.
2. High-Speed Delivery & HR
Rapid introduction of the CRISPR-Cas9/sgRNA and donor components. Cas9 cleavage dramatically increases the rate of Homologous Recombination (HR) event.
3. Selection & Curing
Ultra-rapid selection using the host's fast growth rate to isolate successful integrants. Counter-selection is applied to remove the temporary editing plasmid, ensuring a marker-free strain.
4. Validation & Delivery
Rigorous QC including junction PCR and sequencing of the edited locus to confirm precise, stable, and scarless knock-in. Final validated strain is delivered in pure culture.
Stable Genomic Integration
Achieving permanent, stable integration of large pathways (up to 15kb+) essential for industrial robustness, avoiding all plasmid-related instability issues.
Accelerated Validation
The host's sub-10 minute doubling time allows for rapid verification and functional testing of the integrated pathway, slashing the DBTL cycle time.
Dual Chromosome Strategy
Expertise in leveraging both native chromosomes to achieve optimal gene dosage and co-location of pathway components for balanced expression.
Why is genomic integration better than plasmid expression for V. natriegens?
Genomic integration ensures permanent genetic stability and eliminates the metabolic burden and potential loss of high-copy plasmids, which is crucial for long-term industrial fermentation without antibiotic selection.
What is the maximum size of DNA fragment you can knock-in?
We routinely integrate large biosynthetic fragments up to 15 kb. The size limit is dependent on the complexity and the target site but is significantly larger than typical single-gene insertions.
How do you ensure the integrated gene is highly expressed?
We utilize the host’s native strong promoters or integrate synthetic promoters optimized for V. natriegens expression, along with optimized RBS sequences, to ensure the integrated gene achieves maximal transcription and translation efficiency.
Can you perform knock-in into both chromosomes simultaneously?
Yes. We have specialized strategies to target and integrate genes into specific safe harbor loci on both Chromosome I and Chromosome II, allowing for simultaneous modification or increased gene dosage.
How do you verify a successful knock-in?
We use robust junction PCR (Polymerase Chain Reaction) followed by full Sanger sequencing of the edited genomic locus to confirm the precise, error-free integration of the new DNA and the integrity of the surrounding genome.
What are safe harbor loci in V. natriegens?
Safe harbor loci are non-essential, genetically neutral regions of the genome (on both Chromosomes I and II) identified as optimal sites for foreign DNA insertion. Integration here prevents disruption of essential host genes.
Is the final strain guaranteed to be marker-free?
Absolutely. We employ scarless integration techniques that ensure all selection markers and temporary editing plasmids are removed (cured) from the final strain before delivery.
What is the typical turnaround time for a knock-in project?
Due to the host's fast growth, the process from design to validated, marker-free strain is significantly accelerated, typically taking 5-7 weeks, depending on the complexity of the integrated pathway.
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.
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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.