CD Biosynsis provides a comprehensive suite of Nannochloropsis spp. Gene Knockout Services, designed to accelerate the development of high-performing algal strains for biofuels, nutraceuticals, and carbon sequestration. As an oleaginous microalga capable of accumulating up to 60% of its dry weight in lipids, Nannochloropsis (including N. oceanica, N. gaditana, and N. salina) is a cornerstone of marine synthetic biology. Our platform utilizes precision CRISPR-Cas9 and Cas12a systems to perform targeted gene disruptions, overcoming historical challenges such as low homologous recombination rates and robust epigenetic silencing mechanisms.
Our gene knockout strategies are tailored to the haploid nature of the Nannochloropsis genome, ensuring that modifications instantly manifest their resulting phenotypes. By utilizing DNA-free Ribonucleoprotein (RNP) delivery and markerless episomal systems, we generate "clean" knockout mutants that avoid the permanent integration of foreign DNA or antibiotic markers. This is particularly critical for industrial applications where regulatory compliance and genetic stability are paramount. Whether you are targeting carbon partitioning pathways to enhance lipid titers or knocking out light-harvesting complex genes to reduce antennae size for better light penetration, our services provide verified, monoclonal strains ready for scale-up.
Get a QuoteAchieving stable gene knockouts in Nannochloropsis requires a deep understanding of its unique genetic architecture, characterized by small cell size and a robust cell wall. Our platform leverages advanced electroporation protocols to deliver CRISPR machinery with high efficiency. We specialize in the disruption of primary carbon sinks, such as cellulose (cesA) and chrysolaminarin (storage carbohydrate) biosynthesis, to redirect metabolic flux toward triacylglycerol (TAG) and EPA production. [Image showing the metabolic redirection in Nannochloropsis from starch to lipids via gene knockout]
Our approach integrates predictive metabolic modeling with experimental validation. Before laboratory implementation, we simulate the effects of specific knockouts using genome-scale metabolic models (GEMs) to identify potential growth-productivity trade-offs. This rational design phase is followed by high-throughput screening of mutant libraries using automated FACS systems and lipidomic profiling. By combining these tools, we ensure that the resulting knockout strains exhibit the desired metabolic characteristics without compromising their industrial resilience or photosynthetic efficiency under environmental stress.
We provide multiple pathways for gene disruption, depending on the research objectives and the requirement for transgene-free strains.
Targeted Indels
Utilizing Cas9 or Cas12a to create site-specific double-strand breaks (DSBs), resulting in frameshift mutations and permanent loss-of-function.
Multiplexed KO
Simultaneous disruption of multiple genes in a single transformation, ideal for knocking out redundant gene families like light-harvesting complexes.
RNP Delivery
Direct delivery of Ribonucleoprotein (RNP) complexes via electroporation to achieve "clean" edits without any plasmid or marker integration.
Episomal Curing
Utilizing circular episomal vectors that can be "cured" or removed after the edit, producing marker-free non-transgenic engineered lines.
Insertional Libraries
Generating high-throughput random knockout libraries for target discovery and screening for high-lipid or high-growth mutants (HLMs).
Validation Assays
Secondary screening of leads using GC-MS for fatty acid composition and PAM fluorometry for photosynthetic performance.
Our systematic pipeline ensures the precision of genetic modifications and the genetic stability of the final delivered strains.
1. Computational Design
2. Build & Transformation
3. Monoclonal Selection
4. Stability & Phenotyping
Establishing target gene profiles and bioinformatic gRNA design. Codon optimization of Cas9/Cas12a for Nannochloropsis and off-target risk assessment.
Preparation of RNP complexes or episomal vectors. Transformation of Nannochloropsis cells via optimized electroporation or biolistics.
Verifying genetic stability over 30+ generations. Full physiological characterization, including lipid productivity and growth kinetics under simulated industrial conditions.
Haploid Precision
Efficient targeting of the haploid genome ensures immediate phenotypic expression and simplifies the selection of stable knockout mutants.
Transgene-Free Options
Industry-leading expertise in RNP and episomal methods to deliver strains free of antibiotic markers and foreign DNA.
Industrial Optimization
Screening protocols that evaluate strain performance in high-light, high-CO2, and nutrient-limited environments to ensure industrial readiness.
Full OMICS Support
Integration of transcriptomic and lipidomic data to validate the metabolic impact of every knockout at the systems level.
1. Is Nannochloropsis haploid or diploid?
Most industrial Nannochloropsis strains (like N. oceanica W2J3B) are haploid, which means a single gene disruption is sufficient for a complete knockout phenotype.
2. How do you confirm the knockout has occurred?
We utilize a combination of junction PCR to detect indels and Sanger or Next-Generation Sequencing (NGS) to verify the exact nature of the mutation and the loss of the reading frame.
3. Can you perform knockouts in the chloroplast genome?
Yes, we offer specialized chloroplast engineering via biolistic transformation for genes located in the plastid genome, which is critical for photosynthetic engineering.
4. What are the advantages of RNP-based knockouts?
RNP delivery avoids the risk of random integration of antibiotic resistance markers, making the final strain "non-GMO" in certain regulatory jurisdictions and more stable for long-term production.
5. Which species of Nannochloropsis do you support?
We have established protocols for N. oceanica, N. gaditana, N. salina, and N. oculata. We can also optimize protocols for proprietary industrial isolates.
6. How do you ensure the final strain is monoclonal?
We perform automated single-cell sorting (FACS) followed by multiple rounds of selective plating and genomic verification to ensure the strain originates from a single edited cell.
7. What metabolic targets are commonly knocked out?
Common targets include nitrate reductase (NR) for screening, cellulose synthases to redirect carbon to lipids, and specific lipases or catabolic enzymes to prevent lipid degradation.
8. What is the typical lead time for a knockout strain?
A standard single-gene knockout project typically takes 14 to 18 weeks from design to delivery of the verified monoclonal strain.
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.