Programmable adenine base editing in cyanobacteria using an engineered TadA-Cas9 fusion

Area of Science:

• Microbiology
• Molecular Biology
• Biotechnology

Background:

• Cyanobacteria are vital photosynthetic prokaryotes with significant potential in biomanufacturing and research.
• Advanced genome editing tools, especially CRISPR systems, have faced limitations in cyanobacterial applications.

Purpose of the Study:

• To develop a novel and efficient genome editing tool for cyanobacteria.
• To enable precise A·T to G·C base editing without inducing double-strand breaks or requiring donor templates.

Main Methods:

• Development of pCyABE, an adenine base editor utilizing a TadA-Cas9 nickase fusion.
• Demonstration of editing efficiency in cyanobacterial strains Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120.
• Assessment of multiplex editing capability and off-target activity.

Main Results:

• Achieved high editing efficiency for A·T to G·C conversions in cyanobacteria.
• Demonstrated broad usability across different cyanobacterial species.
• Enabled multiplex editing and start codon disruption for gene function studies.
• Showcased low off-target mutations and effective removal via sucrose counterselection.

Conclusions:

• pCyABE is a versatile and efficient genome editing platform for cyanobacteria.
• This tool significantly expands the genetic engineering capabilities for cyanobacterial research and biotechnology.
• Facilitates precise genetic modifications crucial for advancing cyanobacterial applications.