CD Biosynsis provides professional Nannochloropsis spp. Gene Knock-in Services, offering precise site-specific integration of exogenous genetic material into the genome of this industrially vital microalga. Nannochloropsis species are celebrated for their ability to thrive in various saline environments and produce high concentrations of eicosapentaenoic acid (EPA) and triacylglycerols (TAG). However, traditional random integration often leads to unpredictable expression levels and "position effects." Our services utilize advanced CRISPR-mediated Homology-Directed Repair (HDR) to ensure that your genes of interest—whether they are metabolic enzymes, fluorescent reporters, or therapeutic proteins—are integrated into optimal genomic locations for stable and high-level expression.
Our gene knock-in platform is specifically optimized for the unique biological constraints of Nannochloropsis, including its robust cell wall and highly efficient non-homologous end joining (NHEJ) pathway. By employing specialized donor templates, such as single-stranded DNA (ssDNA) or chemically modified double-stranded DNA (dsDNA), and optimizing the timing of CRISPR machinery delivery, we significantly enhance the frequency of site-specific recombination. We offer comprehensive solutions ranging from the development of "Safe Harbor" integration lines to the construction of multi-gene metabolic pathways. Our goal is to transform Nannochloropsis into a precision-engineered cell factory capable of producing high-value bioproducts with industrial-grade reliability.
Get a QuoteKnocking in genes into Nannochloropsis spp. allows for the expansion of its metabolic repertoire, enabling the production of non-native pigments, specialized lipids, or recombinant proteins. Unlike random insertion, site-specific knock-in ensures that the transgene is placed in a transcriptionally active region without disrupting essential endogenous genes. Our platform identifies and utilizes "Genomic Safe Harbors"—regions where transgene expression is high and stable, and where the integration does not negatively impact the growth or photosynthetic efficiency of the host.
We also specialize in "Scarless" and "Markerless" knock-in techniques. By using Ribonucleoprotein (RNP) complexes and temporary selection markers that can be subsequently removed (e.g., via Cre-Lox or similar systems), we can provide engineered Nannochloropsis strains that are free of antibiotic resistance markers. This is a critical advantage for commercial production and regulatory approval, as it minimizes the risk of horizontal gene transfer in open-pond or large-scale photobioreactor cultivation systems.
Our technical team employs a variety of strategies to achieve precise integration and optimal expression of transgenes.
Predictable Expression
Integration into pre-validated genomic locations to avoid gene silencing and ensure consistent transgene performance across different batches.
Minimal Bio-impact
Ensuring the integration does not interfere with native lipid metabolism or photosynthetic rates, maintaining wild-type growth kinetics.
Fluorescent Tagging
Precise C-terminal or N-terminal tagging of endogenous proteins (e.g., Carbonic Anhydrase) with GFP/YFP to study localization and dynamics.
Affinity Pull-down
Inserting Flag, His, or Myc tags at the native locus to facilitate protein purification and protein-protein interaction studies.
Cassette Integration
Integration of multi-gene metabolic cassettes (up to 5-10 kb) for the production of high-value secondary metabolites.
Promoter Swapping
Replacing native promoters with inducible or stronger constitutive promoters to upregulate specific endogenous metabolic branches.
Our rigorous pipeline ensures the precision of the knock-in event and the high-level performance of the final strain.
1. Design & Vector Build
2. Transformation & HDR Induction
3. Monoclonal Selection
4. Expression Validation
Bioinformatic selection of integration sites and gRNA design. Construction of donor templates with flanking homology arms (HA) optimized for Nannochloropsis codon bias.
Co-delivery of CRISPR machinery (RNPs or plasmids) and donor DNA via optimized high-voltage electroporation. Utilization of small-molecule enhancers to promote HDR over NHEJ.
Confirmation: Verification of the knock-in junction and sequence via NGS. Functional testing: Quantification of mRNA and protein levels (Western Blot/qPCR) and metabolic bioproduct yield.
Precision Placement
Avoid the "position effects" and unpredictable expression of random mutagenesis with site-specific HDR-mediated integration.
Safe Harbor Library
Access to our proprietary library of pre-validated Safe Harbor loci for different Nannochloropsis species to guarantee high expression stability.
Marker-Free Options
Advanced protocols for marker excision or RNP-mediated markerless knock-in to satisfy industrial and regulatory requirements.
High Cargo Capacity
Proven capability to integrate large, complex multi-gene metabolic pathways into the Nannochloropsis genome.
1. What is the typical efficiency of HDR-mediated knock-in in Nannochloropsis?
While native HDR is low, our optimized protocols utilizing RNP delivery and donor design typically yield a sufficient frequency of site-specific clones for robust monoclonal selection.
2. Can you knock in genes into the chloroplast genome?
Yes, we offer specialized chloroplast engineering via biolistic transformation. The chloroplast is ideal for very high-level protein expression and avoids nuclear gene silencing.
3. How do you prevent gene silencing of the integrated transgene?
We use Nannochloropsis-specific promoters, terminators, and full codon optimization. Integration into Safe Harbor loci also significantly reduces the risk of epigenetic silencing.
4. What is the maximum size of a gene cassette you can knock in?
We have successfully integrated cassettes ranging from 2 kb to 8 kb. Larger pathways may require multiple rounds of integration or specialized vector systems.
5. Do you provide validation of protein expression?
Yes, our service includes functional validation via qPCR for transcript levels and Western Blot or LC-MS/MS for protein quantification.
6. Are the delivered strains free of antibiotic markers?
We offer markerless knock-in options using transient selection or marker excision systems (like Cre-Lox) to provide "clean" production strains.
7. How do you verify the knock-in junction?
We perform junction PCR followed by Sanger sequencing of the 5' and 3' integration boundaries. For complex projects, we utilize Whole Genome Sequencing (WGS).
8. What is the turnaround time for a knock-in project?
A standard single-site knock-in project from design to delivery of a verified monoclonal strain typically takes 16 to 22 weeks.
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.