Computational Molecular Biology 2025, Vol.15, No.5, 245-253 http://bioscipublisher.com/index.php/cmb 24 6 unnecessary off-targets, and also affect the feasibility of multi-target editing. With the continuous update of various computing tools and large-scale CRISPR libraries, the accuracy and scalability of tomato genome editing have also improved (Satheesh et al., 2021). This study will focus on discussing these technological advancements, combining their practical applications in tomato breeding and functional genomics. It will also touch upon the existing problems and possible future development directions. 2 Fundamentals of CRISPR Technology in Plants In the field of plant genome editing, CRISPR technology has now almost become a "standard tool". Whether it is Cas9 or Cas12a, essentially they both rely on RNA to lead the way, allowing the enzyme to find the designated DNA position for cleavage (Bandyopadhyay et al., 2020). These operations may sound complicated, but their purpose is actually very direct: to modify genes more quickly and accurately, thereby promoting breeding and gene function research. Especially for crops like tomatoes, to enhance the editing effect, it is inevitable to understand the working mode of CRISPR, the role of guide RNA, and how the editing system enters cells. 2.1 Mechanism of CRISPR-Cas9 and CRISPR-Cas12a systems Both Cas9 and Cas12a belong to Class II CRISPR systems and are widely used in plant research, but their cutting methods are not exactly the same. Cas9 relies on sgRNA to identify the target, and the cut surface is generally flat. Cas12a uses crRNA and will form a sticky misaligned incision (Tang et al., 2019). In addition, Cas12a can handle crRNA arrays by itself, which makes multiple editing more convenient. It recognizes PAM rich in T, while Cas9 prefers sequences rich in G, and the two complement each other perfectly. Some experiments on corn and rice have shown that Cas9 is usually more stable in terms of efficiency and specificity, but Cas12a has its own advantages in multifunctionality and operational flexibility, although it still needs further refinement (Lee et al., 2018). 2.2 Role of guide RNA (gRNA) in target specificity and editing efficiency In the entire editing system, the importance of gRNA is often underestimated. It determines whether nucleases can accurately locate the target site and even affects the final editing efficiency. A well-designed system will have a lower off-target rate and a more stable performance. In recent years, many computational tools have begun to optimize the prediction of gRNA. Coupled with some expression strategies (such as the single transcript unit system), the co-expression of Cas proteins and gRNA has become more reliable (Tan et al., 2024). Especially for crops with relatively complex genomes like tomatoes, the design of grnas is even more demanding; otherwise, it is difficult to achieve sufficient precision in trait improvement. 2.3 Delivery methods of CRISPR components in plant cells No matter how advanced CRISPR is, it must first enter cells to function. There are three commonly used methods in plants: Agrobacterium-mediated, PEG protoplast transfection, and gene gun (Saini et al., 2023). The most common bacterium in tomatoes is Agrobacterium because it is stable, highly efficient and does not insert a large number of copies. PEG and gene guns, on the other hand, are more suitable for editing without exogenous DNA, which can reduce some regulatory concerns. Different delivery methods can affect editing efficiency and tissue specificity, and also determine whether transgenic final plants can be obtained. 3 Overview of CRISPR Design Algorithms When choosing appropriate target sites, researchers now generally rely on various CRISPR design algorithms. Such tools are not designed to make the process overly complicated, but to ensure more accurate editing and fewer off-target editing. They usually identify potential targets based on PAM sequences, and then make further judgments by combining factors such as activity scores and sequence mismatches (Xie et al., 2014). By taking into account both the genomic background and sequence features, these algorithms can help identify more suitable grnas, especially in plants with relatively complex genomes like tomatoes, where this step is particularly crucial. 3.1 Key features of CRISPR design tools Most CRISPR design tools share some common features, such as target screening, off-target prediction, and an interface where users can freely adjust parameters. When screening targets, they would first scan PAM and then
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