Genome Engineering of Phaeodactylum tricornutum

1. Strain Characteristics and Biological Background

As a model species of diatoms, Phaeodactylum tricornutum, after breaking through the limitations of its unique siliceous cell wall structure via gene editing, has become a characteristic strain for functional gene editing and the research and development of new medical active substances. The core applications of gene editing are in the field of high-end biomedicine, specifically including: directional research and development and preparation of new anti-tumor and anti-inflammatory active substances, synthesis of raw materials for precision diagnostic reagents (specific proteins, enzymes), screening of metabolic products related to rare disease treatment. At the same time, it can be used for the innovative research of diatom gene editing technology, adapting to the differentiated R&D needs of high-end scientific research institutions and biopharmaceutical enterprises.

2. Principles and Processes of Gene Editing Technology

The core adopts the CRISPR-Cas9 editing system, combined with cell wall treatment technology and electroporation transformation method, to break through the transformation barrier of diatom siliceous cell walls and achieve precise editing. The specific process is as follows:

(1) Cell wall pretreatment: A special enzymatic hydrolysis system is used to gently remove the siliceous cell wall of Phaeodactylum tricornutum, avoiding damage to cell activity and laying a foundation for transformation;

(2) Target design and sgRNA synthesis: Combined with the strain genome sequence, functional gene targets are accurately designed, and off-target risk sites are ensured through the GeneRulor high-throughput sgRNA screening platform to ensure target specificity and editing efficiency;

(3) Vector construction: Construct a recombinant editing vector suitable for diatom cells, and introduce Cas protein gene and homologous fragments;

(4) Efficient transformation: The optimized electroporation transformation method is adopted to introduce the recombinant vector into pretreated cells, improving transformation efficiency;

(5) Targeted mutagenesis and screening: Induce target gene mutagenesis through directional editing, and quickly screen high-activity metabolite mutant strains relying on the GeneRulor high-throughput screening platform;

(6) Editing verification and off-target detection: Verify editing accuracy through PCR and sequencing, and check off-target risks using GeneRulor's core off-target detection technology;

(7) Strain recovery and propagation: Optimize culture conditions to realize the recovery and stable propagation of edited strains, ensuring the continuous accumulation of active products.

Fig.1 Experimental Process of Phaeodactylum tricornutum Gene Editing

3. Service Types

Focusing on functional editing and high-end needs, we provide differentiated customized services:

(1) Targeted mutagenesis services: Directionally edit functional genes and screen high-activity metabolite mutant strains;

(2) Gene knockout/knock-in: Precisely regulate metabolic pathways to increase the accumulation of new medical active substances;

(3) Specific protein expression: Edit strains to achieve high-efficiency expression of specific proteins and enzymes for precision diagnostic reagents;

(4) Cell wall modification and optimization: Optimize cell wall treatment technology to improve transformation and editing efficiency;

(5) High-end strain customization: Customize functional edited strains according to customers' high-end R&D needs to assist in the research and development of new biopharmaceutical products.

4. Technical Advantages

Relying on the characteristics of diatoms and GeneRulor's core technologies, it forms a differentiated competitive advantage:

(1) Leading cell wall treatment technology: Breaks through the transformation barrier of siliceous cell walls, with a cell wall pretreatment efficiency of more than 90% without damaging cell activity;

(2) Precise targeted mutagenesis: Combined with the high-throughput screening platform, high-activity mutant strains can be quickly screened, and the screening efficiency is increased by more than 50%;

(3) High editing efficiency: The editing efficiency is more than 80%, filling the technical gap in the field of diatom gene editing;

(4) Strong product uniqueness: After editing, new anti-tumor and anti-inflammatory active substances can be produced, adapting to high-end biopharmaceutical R&D;

(5) High degree of customization: Customize exclusive editing schemes according to customers' differentiated needs, adapting to high-end scientific research and product R&D;

(6) High technical barrier: Integrate classic literature technologies and independently optimized schemes to form exclusive core technologies with prominent competitiveness.

5. Project Cycle and Delivery Standards

5.1 Project cycle

(1) Single target editing: 40-60 days;

(2) Targeted mutagenesis and screening: 60-80 days;

(3) Specific protein expression: 40-60 days;

(4) High-end strain customization: 60-80 days.

5.2 Delivery standards

(1) Physical delivery: Pretreatment method, positive edited strains (lyophilized powder + liquid seed solution), high-activity mutant strains (on demand), target products (on demand);

(2) Technical data delivery: Target design report, cell wall pretreatment plan, vector construction map, transformation and screening records, mutant strain activity detection report, off-target detection report, sequencing report, culture and propagation manual;

(3) After-sales guarantee: Provide one-on-one high-end technical guidance, assist customers in carrying out the research and development and application of new active substances, and provide long-term technical support and scheme optimization.

References

[1] Marianne, N., Amit Kumar, S., Marthe C.G., H., Torfinn, S., Atle M., B., & Per, W. (2017). CRISPR/Cas9 Gene Editing in the Marine Diatom Phaeodactylum tricornutum. Bio-protocol, 7(15), e2442. https://doi.org/10.21769/BioProtoc.2442