Cotton Genomics and Genetics 2025, Vol.16, No.6, 269-277 http://cropscipublisher.com/index.php/cgg 275 CRISPR/Cas are not limited to gene knockout or insertion. Fine-tuning of the promoter region directly has gradually become a new strategy. For example, some studies have improved the heat resistance of cotton by weakening the activity of the GhCKI promoter (Li et al., 2025). So, the "operating space" of the promoter is not small. If used well, it can precisely exert force. 7.3 Potential of combining promoter variants with precision breeding technologies In terms of promoters, it is not merely an "auxiliary" regulation. When combined with tools like CRISPR/Cas, the breeding efficiency can be further enhanced by one level. In the past, we always paid more attention to genes themselves, but now we are more inclined to conduct targeted optimization on "regulatory switches". By introducing or editing promoter elements, the target genes can be expressed "just right", neither too much nor too little, especially suitable for those pathways related to adverse adaptation (Shi et al., 2023). Khan et al. (2023) mentioned that modifying promoters with the CRISPR system can not only yield novel alleles but also directly enhance traits such as salt tolerance and drought tolerance. If combined with rapid breeding methods and multi-omics analysis, the entire process will be faster and more accurate. The ultimate outcome is not merely the improvement of a single trait, but rather the possibility of superimposing multiple stress resistances and pushing them together to the practical stage, thus providing more confidence in dealing with complex environments. 8 Conclusion How crucial is promoter variation in the process of cotton coping with stress? In fact, there are already quite a few answers to this question. Representative genes like GhNAC2 and GhHSP70-26, as well as transcription factor families such as ERF, NAC, bZIP, and MYB, contain a large number of cis elements in their promoter regions that respond to hormones and abiotic stresses. Once the environment encounters problems, It can regulate how, when and how much genes are expressed (Guo et al., 2023; Naresh et al., 2024). However, whether there is variation and where it occurs are what truly determine the differences in expression. GWAS and functional verification methods have linked many promoter variations (such as SNPS and indels) to drought resistance, salt tolerance and heat tolerance. The impact of many variations on cotton traits is real. For this reason, promoter engineering, molecular marker selection and other techniques are increasingly being used for cotton improvement. But when it comes to how to proceed next, it's far from the time to "wrap it up". The promoter variations of many cotton germplasm resources remain unclear. Whether there is a real impact on their functions and to what extent, many have not been verified. By incorporating eQTL and GWAS analyses and running them again under different developmental stages and environmental backgrounds, more regulatory factors may be unearthed. At the same time, it is also necessary to clarify the relationship between the changes in the promoter sequence and the binding of transcription factors as well as epigenetic modifications. If this step is not thoroughly understood, the regulatory network will remain a "black box". However, on the other hand, tools like CRISPR/Cas do offer an idea - since there is a way to precisely rewrite the promoter, it might indeed be possible to customize alleles with "novel expression patterns" in the future. Of course, challenges are not absent. Whether promoter variations can be stably inherited and whether they will definitely lead to good outcomes still need to be verified in practice. The multi-gene control and environmental dependence already make stress resistance "elusive". You may move one site, and the result will be a domino effect. Not to mention the difficulties such as the transformation efficiency, expression fluctuations, and network interaction of the promoter sub-project, which are all realistic problems right before us. However, as long as the ideas can be integrated with tools such as precision breeding, rapid breeding, and molecular markers, the breeding efficiency and the implementation speed of stress-resistant varieties are likely to increase significantly. Today, with the increasing pressure of climate change, this path is worth continuing down. Acknowledgments We would like to express our gratitude to the reviewers for their valuable feedback, which helped improve the manuscript. Conflict of Interest Disclosure The authors affirm that this research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest.
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