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ORIGINAL RESEARCH article
Front. Plant Sci.
Sec. Plant Biotechnology
Volume 16 - 2025 | doi: 10.3389/fpls.2025.1540425
Computationally Derived RNA Polymerase III Promoters Enable Maize Genome Editing
Provisionally accepted
Ervin D Nagy 
Ian W Davis 
Shanshan Song Valerie No Chenxi Wu Lisa Kanizay Sarah Turner-Hissong Hong Li 
Xudong Ye Jeffrey C Berry Brandi Chiapelli 
Jennifer PC To 
Matthew S Marengo *- Bayer Crop Science (United States), St. Louis, United States
CRISPR endonucleases require cognate non-coding RNA species for site-specific activity. These RNA species are typically expressed using endogenous RNA polymerase III (Pol III) promoters compatible with the host species. This study describes applications of novel Pol III promoters, which were computationally derived from a training set of monocot U6 and U3 promoters. These promoters enabled genome editing in maize protoplast cells and maize plants. Out of 37 novel promoters, 27 performed similarly to a control U6 promoter. Multiplexing five novel promoters in one construct enabled simultaneous editing of the maize genome at 27 unique sites in a single plant. Moreover, repeating the same CRISPR RNA (crRNA) with multiple novel promoters improved editing up to three-fold at a low-efficiency target site in maize plants. The ability to computationally derive novel Pol III promoters on-demand increases genome editing flexibility and efficiency in maize.
Keywords: RNA Polymerase III, Promoter, CRISPR, LbCas12a, maize ( Zea mays L.), plant
Received: 05 Dec 2024; Accepted: 21 Feb 2025.
Copyright: © 2025 Nagy, Davis, Song, No, Wu, Kanizay, Turner-Hissong, Li, Ye, Berry, Chiapelli, To and Marengo. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence:
Matthew S Marengo, Bayer Crop Science (United States), St. Louis, United States
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