Streamlined and efficient genome editing in Cupriavidus necator H16 using an optimised SIBR-Cas system.

Area of Science:

• Microbiology
• Synthetic Biology
• Biotechnology

Background:

• Cupriavidus necator H16 is a key microbial platform for converting carbon dioxide into valuable products.
• Existing genome editing methods for C. necator are often inefficient and time-consuming, limiting its industrial use.
• Accelerating genome editing is crucial for harnessing C. necator's potential in a bio-based economy.

Purpose of the Study:

• To simplify and expedite the genome editing process in Cupriavidus necator.
• To enhance the efficiency and versatility of genome editing tools for C. necator.
• To facilitate the industrial application of C. necator for CO2 valorisation.

Main Methods:

• Utilized the Self-splicing Intron-Based Riboswitch (SIBR) system to control Cas9 activity.
• Implemented SIBR to delay Cas9-based counterselection, improving editing efficiency.
• Developed SIBR2.0 to regulate Cas12a expression for gene deletion.

Main Results:

• Achieved over 80% editing efficiency at two genomic loci within 48 hours using SIBR and Cas9.
• Demonstrated ~70% editing efficiency for gene deletion using SIBR2.0 and Cas12a.
• Successfully streamlined the genome editing pipeline for C. necator.

Conclusions:

• The SIBR system significantly accelerates and improves genome editing in C. necator.
• SIBR2.0 expands the genome editing toolbox for C. necator, enabling gene deletion.
• These advancements facilitate the industrial application of C. necator for sustainable CO2 valorisation.