Maize Genomics and Genetics 2025, Vol.16, No.6, 294-303 http://cropscipublisher.com/index.php/mgg 296 Figure 1 (A). Total lignin content in WT and mutant lines subjected to both normal and drought treatments; (B). Phenotypic photographs of WT and mutant lines after various treatments, T1 and T3, represent two ZmDIR11-EMS mutant lines with different mutation profiles; Scale bar = 10 cm; (C–G). Growth parameters of WT and mutant lines T1 and T3 under normal and drought conditions, which include above-ground length, root length, fresh weight, dry weight, and chlorophyll content in leaves (from C to G, respectively); (H). Proline (Pro) content in the leaves of WT and mutant lines T1 and T3 under normal and drought treatments. Data are presented as the mean of triplicate values, with error represented as standard deviation (SD). Statistical significance is indicated as non-significant (ns), p < 0.05 (*), andp< 0.01 (**) (Adopted from Zhao et al., 2025) 3 CRISPR/Cas9 as a Genome Editing Tool in Plants 3.1 Mechanism of CRISPR/Cas9: double-strand breaks and repair pathways In plants, the core of gene editing is actually not "cutting", but "repairing". The reason why the CRISPR/Cas9 system is powerful is that it can create a double-strand break (DSB) at a specific position in DNA, and all of this relies on guide RNA (gRNA) to lead the Cas9 enzyme to the target sequence (Bao et al., 2019). Once a break occurs, cells will naturally attempt to repair it. The commonly used methods mainly include two: non-homologous end joining (NHEJ) and homologous recombination (HR). The former is like random mending, prone to errors. Thus, insertion or deletion (indel) has become a common outcome, which is precisely exploited by scientists to "knock out" a certain gene. The latter is more meticulous and requires providing repair templates that can achieve precise rewriting (Xue and Greene, 2021). However, most plant somatic cells prefer to use NHEJ, which makes CRISPR/Cas9 particularly adept at creating loss-of-function mutations. Strictly speaking, it is not a "perfect fix", but rather a natural strategy of "winning by mistake". 3.2 Advantages over traditional breeding and RNA interference (RNAi) To talk about the advantages of CRISPR/Cas9, we first need to look at the limitations of the old methods. Traditional breeding relies on hybridization, screening and backcrossing, which has a long cycle, low efficiency and is also limited by genetic background. CRISPR/Cas9 can precisely "target" and modify, much faster, and can also modify multiple genes at once (Ahmad, 2023). RNA interference (RNAi) may sound like it can also silence genes, but it usually only reduces expression and does not achieve complete knockout. Sometimes, off-target problems may also occur (Chen et al., 2019). In contrast, CRISPR/Cas9 not only has high specificity but also can yield stable and heritable mutant strains. More importantly, some strategies can even leave no trace of exogenous DNA after editing, thus bypassing the "genetically modified" label and making it much easier in terms of regulation and public acceptance. In other words, this technology makes "precision" and "tracelessness" possible.
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