Computationally derived RNA polymerase III promoters enable maize genome editing

Area of Science:

• Plant molecular biology
• Genome editing technologies
• Biotechnology

Background:

• CRISPR endonucleases rely on specific non-coding RNA for targeted DNA modification.
• RNA polymerase III (Pol III) promoters are commonly used for expressing these essential RNA components in various species.
• Developing species-specific promoters is crucial for efficient and precise genome editing.

Purpose of the Study:

• To computationally derive and validate novel Pol III promoters for CRISPR applications in maize.
• To assess the performance of these novel promoters in maize protoplast cells and whole plants.
• To evaluate the potential of these promoters for multiplexed and enhanced genome editing in maize.

Main Methods:

• Computational derivation of novel Pol III promoters from monocot U6 and U3 promoter sequences.
• Testing 37 novel promoters in maize protoplast cells and plants.
• Assessing promoter activity by measuring genome editing efficiency.
• Constructing and testing multiplexed gene editing systems using multiple novel promoters.
• Evaluating the impact of multiple promoters on CRISPR RNA (crRNA) efficiency.

Main Results:

• 27 out of 37 computationally derived promoters demonstrated performance comparable to a control U6 promoter.
• Successful application of novel promoters for genome editing in maize protoplasts and plants.
• Multiplexing five novel promoters enabled simultaneous editing at 27 distinct genomic sites in a single maize plant.
• Using multiple novel promoters with the same crRNA increased editing efficiency up to threefold at a challenging target site.

Conclusions:

• Computationally derived Pol III promoters offer a flexible and efficient tool for CRISPR genome editing in maize.
• These novel promoters facilitate multiplexed editing and enhance editing efficiency, particularly at low-efficiency sites.
• The on-demand generation of Pol III promoters expands the toolkit for advanced plant genome engineering in maize.