Genome Engineering of Synechococcus

1. Strain Characteristics and Biological Background

As a representative strain with high transformation efficiency in the cyanobacteria family, Synechococcus features fast growth rate and flexible metabolic regulation. After gene editing, it can rapidly accumulate target products, with core applications in industrial-scale biopharmaceutical product preparation and scientific research transformation. Specifically, it includes large-scale preparation of medical enzymes (such as thrombolytic enzymes and antioxidant enzymes), directional synthesis of anti-tumor active substances, expression of functional fragments for diagnostic reagents, and it also adapts to the needs of biopharmaceutical industrialization production. It is the preferred strain for small and medium-sized enterprises to realize technological landing, and can also be used for the research on metabolic pathway regulation of prokaryotic algae.

2. Principles and Processes of Gene Editing Technology

The CRISPR-Cas9 editing system is adopted, combined with the optimized homologous recombination strategy. Relying on its high-efficiency transformation characteristics, the editing process is simplified and the efficiency is improved. The specific process is as follows:

(1) Precise target design: Combined with the Synechococcus genome sequence, specific sgRNA is designed, and off-target risk sites are excluded through the Shutong high-throughput screening platform;

(2) Efficient vector construction: The vector backbone is optimized to efficiently integrate sgRNA, Cas protein gene and homologous arm fragments, improving vector transformation efficiency and integration stability;

(3) Rapid transformation: The electroporation transformation method is adopted to adapt to the high-efficiency transformation characteristics of the strain, shorten the transformation time and improve the transformation success rate;

(4) Homologous recombination optimization: The independently optimized homologous recombination strategy is adopted to solve the pain point of low efficiency of large fragment insertion in cyanobacteria;

(5) Positive screening and verification: Positive edited strains are quickly screened through resistance screening, PCR identification and sequencing verification;

(6) Off-target detection: The GeneRulor off-target detection technology is used to confirm the editing accuracy;

(7) Large-scale propagation: The culture conditions are optimized to realize the rapid large-scale culture of edited strains and ensure the efficient accumulation of products.

Fig.1 Synechococcus Gene Editing Strategy

3. Service Types

Focusing on the needs of high-efficiency transformation and industrialization, we provide standardized + customized services:

(1) Rapid gene editing services: Single/multiple target knockout and knock-in with short cycle and high efficiency;

(2) Large fragment foreign gene insertion: Directional insertion of foreign large fragment genes such as medical enzymes and active substance synthesis;

(3) Metabolic pathway optimization: Editing related metabolic genes to increase the accumulation of target products and adapt to large-scale production;

(4) Industrial strain customization: Customize edited strains with high expression and high stability according to customers' production needs;

(5) Scientific research-level rapid editing: Provide rapid editing services for scientific research institutions to help shorten the R&D cycle.

4. Technical Advantages

Relying on the high-efficiency transformation characteristics of the strain and GeneRulor technology optimization, the core advantages are prominent:

(1) High transformation efficiency: The electroporation transformation efficiency is more than 92%, which is far higher than that of similar cyanobacterial strains, reducing screening costs;

(2) Short editing cycle: The editing cycle for a single target is only 7-10 days, and the cycle for large fragment insertion is 12-14 days, which greatly improves R&D and production efficiency;

(3) Strong large fragment insertion ability: Optimized homologous recombination strategy, the insertion efficiency of large fragment foreign genes is more than 88%, adapting to the needs of gene insertion such as medical enzymes;

(4) Fast growth rate: The culture cycle of edited strains is short, which can quickly realize large-scale propagation and reduce industrial production costs;

(5) High stability: The edited strains can be passaged stably for a long time, and the product expression is stable, adapting to the long-term industrial production;

(6) Controllable cost: Supported by standardized processes, the editing and culture costs are greatly reduced, adapting to the needs of small and medium-sized enterprises.

5. Project Cycle and Delivery Standards

5.1 Project cycle

(1) Single target knockout/knock-in: 40-60 days;

(2) Large fragment foreign gene insertion (≤5kb): 60-80 days;

(3) Multi-target editing (2-3 targets): 60-80 days;

(4) Industrial strain customization and propagation: 20-30 days.

5.2 Delivery standards

(1) Physical delivery: Positive edited strains (lyophilized powder + liquid seed solution), large-scale cultures (on demand), crude extract of target products (on demand);

(2) Technical data delivery: Target design report, vector construction map, transformation and screening records, PCR verification report, sequencing report, off-target detection report, large-scale culture plan, product detection report;

(3) After-sales guarantee: Provide technical guidance on large-scale culture, assist customers in solving problems such as strain stability and product accumulation in the production process, and provide technical optimization support.

References

[1] Wendt, K. E., Ungerer, J., Cobb, R. E., Zhao, H., & Pakrasi, H. B. (2016). CRISPR/Cas9 mediated targeted mutagenesis of the fast growing cyanobacterium Synechococcus elongatus UTEX 2973. Microbial Cell Factories, 15(1), 115. https://doi.org/10.1186/s12934-016-0514-7