Genome Engineering of Chlamydomonas reinhardtii

1. Strain Characteristics and Biological Background

As the only eukaryotic model alga capable of efficiently editing three genomes (nuclear, chloroplast, and mitochondrial), Chlamydomonas reinhardtii has become a core research and development carrier in the field of biomedicine, thanks to its advantages of low culture cost and high safety. Its gene editing application scenarios include: high-efficiency expression of therapeutic proteins (antibodies, hormones, vaccines), research and development of gene therapy vectors, functional verification of genes related to genetic disease intervention, preparation of active substances auxiliary to tumor therapy (e.g., antioxidant proteins), and it can also be used for the mechanism research of eukaryotic algal gene editing technologies, adapting to the diverse research and development needs of research institutions and biomedical enterprises.

2. Principle and Process of Gene Editing Technology

The core adopts the CRISPR-Cas9 editing system, combined with dual transformation methods (electroporation and Agrobacterium-mediated transformation), which is compatible with the characteristics of eukaryotic genomes to achieve precise editing of multiple genomes. The workflow is as follows:

(1) Multi-genome target design: Specific sgRNAs are accurately designed for nuclear, chloroplast, or mitochondrial genomes, and target specificity is ensured by combining Shutong's high-throughput screening platform;

(2) Editing vector construction: According to the type of genome to be edited, an appropriate recombinant vector (nuclear genome editing vector, chloroplast-specific vector) is constructed, and Cas protein genes and homologous fragments are introduced;

(3) Strain transformation: Electroporation is preferred (efficient and rapid), and Agrobacterium-mediated transformation is adopted for complex editing needs (to improve integration stability) to achieve efficient vector introduction;

(4) Synchronous multi-target editing (optional): According to customer needs, synchronous editing of multiple genomes (nuclear + chloroplast/mitochondrial) is realized to regulate the high-efficiency expression of target products;

(5) Positive clone screening and verification: Positive strains are screened through resistance screening, fluorescence detection, and PCR verification, and editing accuracy is confirmed by sequencing technology;

(6) Off-target detection: Shutong's core off-target detection technology is used to investigate off-target risks in eukaryotic genomes and ensure editing safety;

(7) Product purification and verification: Therapeutic proteins and other products expressed by edited strains are purified, and their biological activity is verified.

Fig.1 Schematic diagram of CRISPR/Cas9-mediated gene editing in Chlamydomonas reinhardtii

3. Service Types

Relying on its "all-round editing" characteristics, comprehensive scenario-based customized services are provided:

(1) Multi-genome editing service: Independent/synchronous editing of nuclear genome, chloroplast genome, and mitochondrial genome;

(2) Therapeutic protein expression service: Directional editing of strains to achieve high-efficiency expression and purification of exogenous therapeutic proteins (antibodies, vaccines, etc.);

(3) Gene knock-out/knock-in/point mutation: Precise regulation of target genes to verify gene functions or modify metabolic pathways;

(4) Gene therapy vector customization: Modification of Chlamydomonas reinhardtii genome to construct safe and efficient gene therapy delivery vectors;

(5) Research-grade strain modification: Provision of customized edited strains for research institutions, used for gene function research and technology development.

4. Technical Advantages

Combining the characteristics of eukaryotic algae and Shutong's technical advantages, the core highlights are remarkable:

(1) All-round editing capability: The only eukaryotic alga capable of efficient editing of multiple genomes (nuclear, chloroplast, and mitochondrial), adapting to diverse needs;

(2) High editing efficiency: Leading editing efficiency among eukaryotic algae, with nuclear genome editing efficiency reaching over 85% and chloroplast editing efficiency reaching over 80%;

(3) High product activity: Chloroplast editing achieves high-efficiency expression of therapeutic proteins, and the products have natural biological activity without complex in vitro modification;

(4) High safety: The strain itself is non-pathogenic, and there are no harmful metabolites after editing, making it suitable for applications in the medical field;

(5) Standardization and scale: The editing system has been standardized, which can quickly respond to customers' batch sample editing needs;

(6) Strong literature support: Based on the technologies of classic literatures such as Nature Methods and Plant Physiology, the editing system is mature and stable.

5. Project Cycle and Delivery Standards

5.1 Project cycle: Flexibly adjusted according to editing complexity

(1) Single target editing of a single genome: 40-60 days;

(2) Synchronous editing of multiple genomes (2 genomes): 60-80 days;

(3) Therapeutic protein expression and purification: 40-60 days;

(4) Gene therapy vector customization: 20-30 days.

5.2 Delivery standards

(1) Physical delivery: Positive edited strains (lyophilized powder + liquid seed solution), purified products (as required);

(2) Technical data delivery: Target design report, vector construction map, transformation and screening records, multi-genome editing verification report, off-target detection report, sequencing report, product activity detection report, culture and purification manual;

(3) After-sales guarantee: Provision of technical training and one-on-one guidance, assistance to customers in conducting subsequent product applications and strain expansion, and provision of technical support for 3 months.

References

[1] Dokane, D., Bhadra, B., & Dasgupta, S. (2020). CRISPR based targeted genome editing of Chlamydomonas reinhardtii using programmed Cas9-gRNA ribonucleoprotein. Molecular Biology Reports, https://doi.org/10.1007/s11033-020-05922-5.