Maize Genomics and Genetics 2025, Vol.16, No.6, 294-303 http://cropscipublisher.com/index.php/mgg 297 3.3 Transformation methods in maize: agrobacterium-mediated, biolistic, and RNP delivery For CRISPR/Cas9 to truly function in corn, the key lies not in the "scissors", but in "putting the scissors in". There are three common practices, each with its own advantages and disadvantages. Agrobacterium-mediated transformation is the most common, which integrates the CRISPR/Cas9 DNA construct into the plant genome by means of Agrobacterium rhizocarpus. This approach is mature and reliable, but it is not very friendly to certain genotypes of corn, and the insertion position is often random (Li et al., 2021). Another method is the gene gun technique, which uses high-speed particles to directly inject DNA or Cas9-gRNA ribonucleoprotein (RNP) into cells. This method is particularly suitable for monocotyledonous plants like corn, and it can also avoid introducing exogenous DNA when using RNP, achieving true "non-GMO" editing. The third type-RNP delivery-is actually an extension of the gene gun method: Cas9 and gRNA are pre-assembled and then delivered into cells, eliminating the transcription and translation process, improving efficiency, and reducing the risk of off-target. Now it has been proven to be successfully applied in corn, being both clean and efficient. 4 Identification and Selection of Drought-Sensitive Genes 4.1 Omics-based screening of candidate genes under drought stress In corn research, it is not easy to find truly "effective" drought-resistant genes. Scientists often have to dig for clues bit by bit from vast amounts of data. High-throughput technologies such as transcriptome, metabolome and genome-wide association studies (GWAS) are precisely the main tools of this "journey to search for genes". Under drought stress, the transcriptional levels of corn of different genotypes changed astonishingly-thousands of differentially expressed genes (DEGs) were detected, which were involved in signal transduction, antioxidation, hormone synthesis and even metabolic regulation (Zenda et al., 2019; Waititu et al., 2021). Metabolomics fills in another piece of the puzzle. Some metabolites, such as molecules involved in tryptophan or amino acid metabolism, have been found to be closely related to drought resistance performance (Li et al., 2024). Meanwhile, GWAS and quantitative trait loci (QTL) analyses also identified several key regions, revealing genes related to differences in drought tolerance-including transcription factors and signaling pathway components (Wu et al., 2021). These results collectively indicate that the drought resistance of corn is the product of a multi-level network rather than a story dominated by a single gene. 4.2 Functional studies validating gene roles in drought responses The candidate genes have been identified. The next step is to confirm that they are "really useful". Researchers usually verify gene functions by means of overexpression, knockout or transgenesis. Taking ZmPP2C15 and ZmNAC111 as examples, the CRISPR/Cas9 tool helps researchers directly observe their "role changes" in drought response (Pang et al., 2024). When positive regulatory factors are overexpressed, corn becomes more resilient-the activity of antioxidant enzymes increases, osmotic protective substances rise, and water use efficiency is also better. Conversely, once those negative regulatory factors or drought-sensitive genes are knocked out, drought resistance can also be improved (Liu et al., 2022). In other words, enhancing drought tolerance is not solely dependent on "strengthening"; sometimes, "weakening" certain genes can be more effective. These experiments have provided empirical evidence for the theory and also offered conclusive evidence for understanding the drought adaptation mechanism of corn. 4.3 Notable drought-sensitive genes targeted in maize: ZmNAC111, ZmPP2C-A10, ZmDREB2A Among the numerous candidate genes, ZmNAC111, ZmPP2C-A10 and ZmDREB2A frequently appear in research reports and have also become key targets for gene editing. ZmNAC111 is a transcription factor of the NAC family, and its expression is inhibited by the insertion of MITE elements in the promoter. If it is allowed to overexpress, the water use efficiency and drought tolerance of corn will be significantly improved. ZmPP2C-A10 belongs to Class A PP2C phosphatase, but its function is exactly the opposite-it inhibits drought tolerance response. Research has found that reducing or knocking out this gene can enhance drought resistance by regulating ABA signaling and reducing water loss.
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