CD Biosynsis offers high-precision Phaeodactylum tricornutum Gene Knock-in Services, providing a robust platform for the site-specific integration of exogenous DNA into the genome of this industrially significant pennate diatom. Unlike traditional random integration, which often leads to unpredictable expression levels and "position effects" due to the host's complex chromatin environment, our knock-in technology utilizes CRISPR-mediated Homology-Directed Repair (HDR). This ensures that transgenes are inserted into pre-validated genomic "safe harbors" or specific endogenous loci, allowing for stable, high-level expression of metabolic pathways, reporters, or therapeutic proteins.
Our service is specifically engineered to address the unique biological constraints of Phaeodactylum tricornutum, such as its diploid nature and the historically low frequency of homologous recombination in diatoms. By employing advanced donor template designs—including long homology arms and optimized algal regulatory elements—we facilitate the seamless integration of large genetic cassettes. Whether you are aiming to "humanize" glycosylation pathways, assemble multi-enzyme biosynthetic routes for Omega-3 fatty acids, or introduce fluorescent tags for live-cell imaging, our end-to-end solutions provide the technical expertise and analytical verification necessary to achieve your research and production goals.
Get a QuoteAchieving predictable transgene expression in Phaeodactylum tricornutum has traditionally been a bottleneck in diatom biotechnology. Randomly integrated DNA is frequently subjected to transcriptional silencing or variable expression depending on the local genomic context. Our CRISPR-Cas9/HDR-mediated knock-in platform solves these issues by directing the integration of your Gene of Interest (GOI) to specific, well-characterized loci. This precision allows for the maintainance of consistent expression across multiple generations, which is critical for industrial scale-up in photobioreactors.
Our platform is meticulously optimized for the diatom's nuclear environment. We utilize proprietary codon-optimization matrices to ensure that both the CRISPR machinery and the integrated transgenes are translated efficiently despite the specific codon usage of P. tricornutum. By integrating your pathways into "Safe Harbor" sites—genomic regions where insertion does not disrupt essential genes—we ensure that the engineered strains retain their native growth vigor and photosynthetic efficiency while performing their new metabolic functions.
We provide a variety of knock-in strategies tailored to the specific functional requirements of your project, from simple protein tagging to complex pathway assembly.
Locus Selection
Targeting validated intergenic regions or non-essential loci that support high transcriptional activity and genetic stability without affecting host fitness.
Stable Expression
Ensuring that integrated cassettes are protected from the epigenetic silencing mechanisms that typically target randomly integrated DNA in diatoms.
Fluorescent Fusions
Site-specific insertion of fluorescent proteins (e.g., mCherry, GFP) at the C- or N-terminus of endogenous genes to study protein localization and dynamics under native regulation.
Affinity Tags
Insertion of FLAG, HA, or His tags to facilitate protein purification and the study of protein-protein interactions within the diatom cellular environment.
Multi-Gene KI
Simultaneous integration of multiple enzymes to create new biosynthetic routes, such as the synthesis of long-chain polyunsaturated fatty acids (LC-PUFAs).
Regulatory Tuning
Utilizing a library of diatom-specific promoters and terminators to fine-tune the expression levels of each gene within the integrated pathway.
Our rigorous workflow ensures the precise delivery of engineered strains with full genomic transparency and functional verification.
1. Computational Design
2. Build & Preparation
3. Transformation & Enrichment
4. Verification & Delivery
Selection of the optimal integration site and bioinformatic gRNA design. Full codon optimization of the transgene sequence to match the P. tricornutum nuclear bias (approx. 50-55% GC in coding regions).
Synthesis of HDR donor templates with high-fidelity homology arms. Preparation of Cas9 RNPs or episomal vectors. For large inserts, we optimize donor concentration to maximize HDR efficiency.
Genotyping: Confirmation of site-specific integration via 5' and 3' junction PCR and Sanger/NGS sequencing. Phenotypic Characterization: Western blot or metabolic profiling (GC-MS) to confirm functional expression. Delivery of cryopreserved strains.
Position-Effect Elimination
By targeting specific loci, we avoid the unpredictable "random integration" outcomes, ensuring high batch-to-batch consistency in transgene expression.
Large Cassette Capacity
Our platform is capable of integrating large multi-gene metabolic pathways (up to 10 kb+), enabling complex synthetic biology projects in diatoms.
Optimized Regulatory Parts
Access to a comprehensive library of native diatom promoters (e.g., FCP, NR) and terminators to ensure maximal translational throughput.
Genomic Stability
Rigorous stability trials over 50 passages ensure that the integrated DNA remains intact and active, even in industrial-scale cultivation conditions.
1. Why is CRISPR-mediated knock-in better than random integration for diatoms?
Random integration in P. tricornutum often leads to gene silencing or variable expression. CRISPR-mediated knock-in ensures the gene is placed in a "Safe Harbor" locus where it can be expressed stably and predictably.
2. How do you handle the diploid nature of Phaeodactylum tricornutum?
For most applications, mono-allelic integration is sufficient for expression. However, we can perform targeted screening to identify homozygous (bi-allelic) knock-in clones if required for your experimental design.
3. What is the typical size limit for a gene knock-in?
While smaller inserts are more efficient, we have successfully integrated cassettes up to 8-10 kb in size using optimized donor templates and high-efficiency transformation methods.
4. Do you provide codon optimization for the transgenes?
Yes, all transgenes are codon-optimized using our proprietary Phaeodactylum-specific bias matrix to ensure maximal protein expression and avoid mRNA instability.
5. How do you verify the site-specific integration?
We perform "junction PCR" using primers that span the border between the host genome and the donor DNA at both the 5' and 3' ends. This is followed by Sanger or NGS sequencing for definitive proof.
6. Can you perform knock-in in the chloroplast genome?
Yes, we offer specialized chloroplast transformation services via biolistic bombardment to target the polyploid plastid genome for high-level protein accumulation.
7. Are the engineered strains marker-free?
We can utilize "scarless" methods or selection markers that can be cured via episomal removal to provide strains that are free of antibiotic resistance genes.
8. What is the typical turnaround time for a knock-in strain?
A standard knock-in project from computational design to the delivery of a verified monoclonal strain typically takes between 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.