Genome Engineering of Synechocystis

1. Strain Characteristics and Biological Background

As a pivotal model organism for prokaryotic algal gene editing, Synechocystis sp. PCC 6803 exhibits efficient metabolic pathway regulation capabilities following gene editing, and is extensively utilized in the fields of biomedicine, medical excipients, and precision diagnosis. Its core applications include: large-scale preparation of antioxidant components (carotenoids) associated with auxiliary tumor therapy, directed synthesis of medical-grade enzymes (e.g., antioxidant enzymes), high-efficiency expression of functional proteins for diagnostic reagents, as well as serving as a model strain for the research and development of gene editing technologies, thereby facilitating research institutions in conducting studies on the mechanisms of prokaryotic algal gene regulation.

2. Principle and Process of Gene Editing Technology

The core of the technology employs the CRISPR-Cas9/Cas12a editing system integrated with homologous recombination technology, which is compatible with the clear genetic background of Synechocystis sp. PCC 6803 and features a standardized, replicable workflow:

(1) Target design and sgRNA synthesis: Specific sgRNAs are accurately designed based on the strain's genome sequence, and targets with the lowest off-target rate are screened using GeneRulor's high-throughput sgRNA screening platform;

(2) Vector construction: sgRNA, Cas protein genes, and homology arm fragments (knock-in/knock-out fragments) are constructed into an appropriate vector to ensure efficient vector transformation;

(3) Strain transformation: The recombinant vector is introduced into Synechocystis cells via electroporation to achieve efficient vector integration;

(4) Positive clone screening: Successfully edited positive strains are screened through resistance screening combined with PCR verification;

(5) Off-target detection and verification: GeneRulor's core off-target detection technology is utilized to confirm the absence of off-target risks, and editing accuracy is verified through sequencing;

(6) Large-scale culture: Culture conditions are optimized to realize large-scale expansion of positive strains and ensure stable accumulation of target products.

Fig.1 Schematic diagram of CRISPR/Cas9-mediated gene editing in Synechocystis sp. PCC 6803

3. Service Types

Focusing on the core needs of customers, comprehensive customized services are provided throughout the entire process, including:

(1) Gene knock-out service: Directional knock-out of irrelevant metabolic genes to enhance the accumulation of target products;

(2) Gene knock-in service: Directional knock-in of exogenous functional genes (e.g., medical enzyme genes, active substance synthesis genes);

(3) Multigene regulation service: Batch editing of multiple targets to achieve precise regulation of metabolic pathways;

(4) Strain modification and optimization: Secondary optimization of existing edited strains to improve product expression levels and stability;

(5) Research-grade/industrial-grade strain customization: Customization of exclusive edited strains in accordance with customers' research and development or production requirements.

4. Technical Advantages

Leveraging the characteristics of the strain and GeneRulor's technical accumulation, the core advantages are prominent as follows:

(1) High editing efficiency: Combined with the optimized CRISPR-Cas12a system, the editing efficiency can reach over 95%, which is significantly higher than the industry average;

(2) Extremely low off-target rate: Equipped with GeneRulor's independent off-target detection technology, it can accurately detect genome-wide off-target sites to ensure the safety of gene editing;

(3) Short project cycle: With the support of standardized workflows, the editing cycle for a single target ranges from 40 to 60 days;

(4) High transformation efficiency: Adapted to electroporation transformation technology, the strain transformation efficiency exceeds 90%, thereby reducing screening costs;

(5) Strong scalability: Edited strains can rapidly achieve large-scale culture, meeting the requirements of industrial production;

(6) Mature technology: Based on the technical achievements published in classic journals such as Nature Communications and ACS Synthetic Biology, the editing system has undergone long-term verification and exhibits high stability.

5. Project Cycle and Delivery Standards

5.1 Project cycle: Flexibly adjusted according to the type of service

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

(2) Multigene regulation (2-3 targets): 60-80 days;

(3) Strain customization and optimization: 60-80 days;

(4) Large-scale culture service: 20-30 days depending on the culture volume (which can be carried out synchronously with the editing service).

5.2 Delivery standards

(1) Physical delivery: Positive strains (lyophilized powder + liquid seed solution) and large-scale cultures (as required);

(2) Technical data delivery: Target design report, sgRNA sequence, vector construction map, transformation and screening records, PCR verification report, off-target detection report, sequencing report, and culture condition manual;

(3) After-sales support: Provision of one-on-one technical guidance, response to questions related to strain culture and subsequent application of gene editing, and provision of secondary optimization support when necessary.

References

[1] Cengic, I., Canadas, I. C., Minton, N. P., & Hudson, E. P. (2022). Inducible CRISPR/Cas9 Allows for Multiplexed and Rapidly Segregated Single-Target Genome Editing in Synechocystis Sp. PCC 6803. ACS Synthetic Biology, 11(10), 3100-3113.