Repurposing endogenous Type I-D CRISPR-Cas system for genome editing in Synechococcus sp. PCC7002

Area of Science:

• Synthetic biology
• Microbial biotechnology
• CRISPR-Cas gene editing

Background:

• Synechococcus sp. PCC7002 is a promising photosynthetic chassis for CO2 conversion.
• Existing genetic manipulation methods are insufficient for extensive modifications in this cyanobacterium.

Purpose of the Study:

• To develop a novel genome editing tool for Synechococcus sp. PCC7002.
• To overcome limitations of conventional genetic engineering techniques in this strain.

Main Methods:

• Leveraged the endogenous type I-D CRISPR-Cas system of S. PCC7002.
• Developed and applied a novel genome editing tool for targeted gene knockout and DNA fragment deletion.

Main Results:

• Successfully deleted glgA1 and glgA2 genes, creating a double mutant.
• Knocked out large DNA fragments including type I-A (14 kb) and III-B (21 kb) CRISPR-Cas systems.
• Cured the endogenous pAQ5 plasmid (38 kb) from the S. PCC7002 genome.

Conclusions:

• Demonstrated the efficacy of the endogenous CRISPR-Cas system for genome editing in S. PCC7002.
• Established a robust genetic tool for enhancing the strain's potential as a photosynthetic cell factory.
• Provided a foundation for future metabolic engineering efforts to improve biosynthetic efficiency.