Maize Genomics and Genetics 2025, Vol.16, No.4, 229-238 http://cropscipublisher.com/index.php/mgg 232 4 Application Strategies for Base Editing in Maize 4.1 Selection of suitable base editor variants for monocots Not all base editing systems are suitable for monocotyledonous plants like corn, and choosing the right tool is crucial. For instance, when Cas9 is fused with cytidine deaminase, the C in the corn gene can be transformed into T, and the editing efficiency is not low. In some experiments, the editing rate of the plants can reach 43.5% (Zong et al., 2017). Sometimes, to expand the target site range, variants with less stringent PAM requirements, such as xCas9 or Cas9-NG, are also selected, although the hit rate may vary depending on the target site (Fierlej et al., 2022). Moreover, for those stubborn corn varieties with very low conversion efficiency, there is not a complete solution. Researchers found that adding some developmental regulatory factors, such as BABY BOOM, WUSCHEL or GRF-GIF fusion protein, to the transformation vector could significantly increase their transformation and regeneration rates (Zhang et al., 2019a). 4.2 Optimization of delivery systems (e.g., Agrobacterium, Biolistics) To make the editing system truly effective, having editing tools alone is not enough; the key lies in how to incorporate them. The most commonly used methods are still Agrobacterium and gene guns. The transformation efficiency of Agrobacterium was not high before, especially for some difficult-to-transform genotypes. However, with the advent of ternary vectors and genes that regulate morphology, the efficiency in this regard has been enhanced (Vandeputte et al., 2024). There have also been new advancements in gene guns. For instance, by using mRNA or RNP complexes, editing can be accomplished without leaving exogenous DNA (Svitashev et al., 2016; Qiu et al., 2025). There are even faster methods, such as using viral vectors - like barley striped Mosaic virus - to deliver guide RNA. This approach is fast and can target multiple sites at once (Hu et al., 2019). Some studies even bypassed tissue culture and directly edited in superior materials, such as using haploid induction (HI-Edit/IMGE) methods (Kelliher et al., 2019). Of course, nanotechnology is also being experimented with now, such as nanoparticles and cell-penetrating peptides, which might come in handy in the future (Li et al., 2025). 4.3 Validation of edits through molecular and phenotypic screening The editing is done. Whether it will succeed or not still needs to be verified. On the one hand, it can be viewed at the molecular level. For instance, PCR and next-generation sequencing (NGS) can be combined with bioinformatics processes to determine whether the bases have changed or gone off-target (Char et al., 2016). On the other hand, it is also necessary to observe the actual plants that grow, such as testing their herbicide resistance or other agronomic traits to see if they meet expectations. Before stable transformation, it is actually possible to conduct a quick test using the protoplast system to see how efficient it is (Fierlej et al., 2022). The final step is very important: screen out those individuals containing exogenous genes or selection markers, and through genetic separation, leave clean edited plants. This is quite crucial for compliance with regulations and public acceptance (Yamada et al., 2024). 5 Case Study 5.1 Background of ALS gene function and its role in herbicide sensitivity The ALS gene, in the final analysis, its primary function is to enable plants to synthesize several important amino acids, such as valine, leucine and isoleucine. And the first step of this process is accomplished by an enzyme called acetyllactate synthase (also known as acetylhydroxy acid synthase). However, the problem lies precisely here. Some common herbicides on the market-such as sulfonylureas and imidazolinones-are specifically targeted at it. These drugs can interfere with the function of ALS. Plants cannot synthesize amino acids and thus cannot grow normally, and may eventually die directly. However, scientists have found that if the ALS gene undergoes a minor mutation, especially in the part that affects the herbicide binding ability, it can resist these drugs, while the growth of the plant itself is basically unaffected (Li et al., 2020). 5.2 Experimental base editing approaches targeting ALS in maize Recently, an experiment attempted to use the CRISPR/Cas9 system, in combination with a cytidine deaminase and UGI protein, to change certain bases in the two ALS genes of corn - ZmALS1 and ZmALS2 - from C to T. At first, they tested it in corn protoplasts, and later they also used these systems to work on regenerated plants. The result
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