Abstract
Drought stress represents one of the most critical environmental constraints limiting global maize (Zea mays) productivity. While conventional transgenesis and knockout approaches have improved abiotic stress tolerance, they frequently incur pleiotropic growth penalties or regulatory bottlenecks. In this study, we developed an engineered plant-adapted CRISPR-Cas12b base editing system to fine-tune transcriptional responses through precise nucleotide substitutions within cis-regulatory promoter regions. Utilizing an engineered Alicyclobacillus acidoterrestris Cas12b (AacCas12b) cytidine base editor (CBE) targeting T-rich protospacer adjacent motif (PAM) sites, we modified negative regulatory repressor elements within the promoter of ZmDREB1A, a master transcriptional regulator of drought response. Targeted C-to-T substitutions within the repressor binding motifs resulted in editing efficiencies ranging from 31.4% to 46.8% in regenerated T0 maize lines, with minimal bystander indels and negligible off-target activity across the genome. T2 homozygous mutant lines exhibited hyper-inducible expression of ZmDREB1A under drought conditions without altering basal expression during vegetative growth. Consequently, edited plants displayed enhanced relative water content, elevated proline accumulation, reduced malondialdehyde levels, and superior photosynthetic stability during severe water deficit, yielding a 21.6% increase in grain recovery over wild-type controls. Our findings validate Cas12b-mediated promoter base editing as a robust, transgene-free-compatible strategy for rational transcriptional rewiring, offering a sustainable platform for engineering climate-resilient cereal crops.
