Maize Genomics and Genetics 2025, Vol.16, No.6, 294-303 http://cropscipublisher.com/index.php/mgg 300 Similar results have also been repeatedly verified in crops such as rice and wheat-whether overexpressing positive regulatory genes or knocking out negative regulatory factors, it can increase chlorophyll content, reduce stomatal conductance, and ultimately improve survival rate (Usman et al., 2020; Abdallah et al., 2022). In other words, different crops, in different ways, reached similar physiological outcomes. 6.3 Stable yield performance under moderate to severe drought stress The real test is still the output. No matter how well the laboratory performs, the key lies in whether the field can "hold up". Multiple field and controlled environment trials have shown that gene-edited corn yields are more stable and even slightly increase under drought conditions. For instance, the ARGOS8 variant showed a significant increase in grain yield under drought conditions during the flowering period, while no reduction occurred when there was sufficient water (Rai et al., 2023). Similarly, corn with DSD1/ZmICEb and ZmHDT103 knocked out also reduced yield loss. These results indicate that gene knockout is not merely an experimental phenomenon but a strategy that can be translated into actual agronomic advantages. Overall, these studies reveal a trend-through precise gene editing, corn is expected to achieve "stable production without reduction" in water-scarce environments, which is of great significance to agriculture in areas frequently hit by droughts. 7 Challenges and Considerations in CRISPR-Based Drought Tolerance 7.1 Off-target effects and their detection and mitigation In gene editing, what often worries researchers the most is not "unable to cut", but "cutting in the wrong place". The off-target problem of CRISPR/Cas9 is just like this-when the guide RNA is too similar to other genomic sequences, unexpected mutations may occur. However, the good news is that in a complex genome like that of corn, as long as the guide RNA is properly designed and has at least three base misaligns with non-target regions, especially in the seed region near the PAM, the risk of off-target is usually extremely low (Young et al., 2019). There are also more and more methods for detecting such unexpected edits. Computational prediction tools, whole-genome testing (such as CLEAVE-Seq), and direct validation in plants can all help scientists "identify" these minor biases (Erdogan et al., 2023). In addition, high-fidelity Cas9 variants, shorter grnas, and the delivery method in the form of RNP have also been proven to effectively enhance specificity. Overall, these improvements often result in less off-target variation than natural mutations in traditional breeding. In other words, this "precision revolution" is more controllable than people imagine. 7.2 Regulatory landscape and public acceptance of gene-edited crops A scientific breakthrough does not mean that one can immediately go to the fields. The regulation and public acceptance of CRISPR-edited crops remain the key factors determining their fate. The attitude in the United States is relatively open-if exogenous DNA is not introduced into plants, they are often not classified as strictly genetically modified (Ahmar et al., 2023), which has accelerated the pace of commercialization. But not all countries are so optimistic. In regions such as the European Union and New Zealand, gene-edited crops are still treated as genetically modified, with slow approval and numerous restrictions (Ansari et al., 2020). The public's attitude is also complex: what they care about is not the technical principle, but safety, transparency, and "whether it is genetically modified or not". Therefore, using RNP delivery to obtain edited corn without exogenous fragments might alleviate some concerns and bring science closer to society. 7.3 Integration with breeding programs and scaling for field use The maturity of technology does not mean that it can be immediately transformed into breeding achievements. Integrating CRISPR/Cas9 into the traditional breeding system requires more collaboration and patience. Take multi-genome editing strategies such as BREEDIT as an example. They can simultaneously modify multiple genes, accelerate trait improvement, and also help screen out the optimal drought-resistant gene combination (Shelake et al., 2022). However, problems also arise: corn of different genotypes show significant differences in transformation and regeneration, and the editing efficiency varies. Some laboratory achievements fail to adapt to the local
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