Cotton Genomics and Genetics 2025, Vol.16, No.6, 300-309 http://cropscipublisher.com/index.php/cgg 302 3 Genome Editing Tools for Precision Breeding 3.1 CRISPR-Cas systems in cotton Among various breeding techniques, CRISPR/Cas9 has almost become a "regular guest". Researchers prefer it not only because of its simple operation and high efficiency, but also because it can simultaneously trigger targeted mutations of homologous genes in allotetraploid cotton. In other words, several related genes can be modified at one time without having to be dealt with separately. What it can do is far more than just "knocking out" a certain gene. Through CRISPR/Cas9, people can achieve gene knockout, fragment deletion, and even multiple editing, with several modifications carried out simultaneously in a single plant. In recent years, "new members" such as Cas12a (Cpf1) and Cas12b have also been successfully introduced into cotton research (Figure 1) (Wang et al., 2020). They are more flexible in identifying targets, have a lower risk of missing the target, and also have higher editing efficiency. The result is that these genetic modifications can often be stably inherited, and the mutations they produce can also be carried on to the next generation, providing a more controllable approach for the improvement of cotton traits. Figure 1 Vector, sgRNAs map, genetic transformation and generated plant through AacCas12b system in cotton. (a) Schematic view of sgRNA1 and sgRNA2 target sites in the GhCLA gene. The target sequences are highlighted in blue, and the PAM sites are highlighted in red. (b) Schematic of the T-DNA region of GhRCas12b vector. (c) The Agrobacterium-mediated genetic transformation and plant regeneration of transgenic plants. (c-A) Co-culture stage. (c-B-C) Callus induction and differentiation. (c-D) Somatic embryogenesis. (c-E) Plant regeneration. (c-F) The acclimatization of regenerated plant in nutrient solution. (c-G-H) Transgenic plants grown in the greenhouse. (d) Total number of T0 generated plants and the number of positive edits produced under different temperature conditions (Adopted from Wang et al., 2020) 3.2 Delivery methods and transformation protocols Although gene editing sounds advanced, the step of "delivering the tools into the cells" is not easy. The transformation of cotton has always been a headache for researchers, as it is time-consuming and unstable. The commonly used methods nowadays are still Agrobacterium-mediated transformation or gene gun bombardment, but both of these approaches have their limitations. To bypass these obstacles, some new solutions have been proposed. For instance, apical meristem transformation (SAMT) and virus-induced genome editing (VIGE) have significantly improved editing efficiency and can also overcome genotype-dependent issues (Lei et al., 2022). In addition, before formal stable transformation, transient expression systems and hair-like root transformation are often used to test the effectiveness of sgRNA (Zhou et al., 2022). Doing so can help detect "invalid designs" in advance and save a lot of time. As for the expression optimization of sgRNA, researchers found that using the
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