Computational Molecular Biology 2025, Vol.15, No.5, 245-253 http://bioscipublisher.com/index.php/cmb 24 5 Case Study Open Access Case Study: Application of CRISPR Design Algorithms in Tomato Genome Editing Dandan Huang Hainan Institute of Biotechnology, Haikou, 570206, Hainan, China Corresponding author: dandan.huang@hibio.org Computational Molecular Biology, 2025, Vol.15, No.5 doi: 10.5376/cmb.2025.15.0024 Received: 07 Aug., 2025 Accepted: 18 Sep., 2025 Published: 09 Oct., 2025 Copyright © 2025 Huang, This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.6 Preferred citation for this article: Huang D.D., 2025, Case study: application of CRISPR design algorithms in tomato genome editing, Computational Molecular Biology, 15(5): 245-253 (doi: 10.5376/cmb.2025.15.0024) Abstract CRISPR-Cas systems have revolutionized plant genome editing by enabling precise, efficient, and targeted modifications, with tomato (Solanum lycopersicum) serving as a key model for translational applications in crop improvement. This study explored the application of CRISPR design algorithms in the selection and validation of guide RNAs (gRNAs) for optimizing tomato genome editing. The mechanisms of action of the CRISPR-Cas9 and Cas12a systems were analyzed, and major design tools such as CHOPCHOP, CRISPOR, and CRISPR-P were compared. The performance of the algorithms was evaluated in terms of targeting efficiency and specificity. Using the SlMLO1 gene as a functional target, this study conducted a case study using CRISPR-P and CRISPR-GE platforms to design and evaluate gRNAs, achieving effective gene editing and enhancing tomato resistance to powdery mildew. This study highlights the challenges faced by algorithms in predicting off-target effects and adapting to the complex tomato genome, while also emphasizing the potential of integrating machine learning and tomato-specific databases to improve design accuracy. This research underscores the increasing synergy between computational biology and experimental biology, paving the way for the development of next-generation AI-assisted genome editing tools suitable for crops. Keywords CRISPR-Cas9; Guide RNA design; Tomato genome editing; Algorithm optimization; SlMLO1 gene 1 Introduction In the field of plant genome editing, the CRISPR-Cas system is now almost universally mentioned, but its true impact often cannot be simply summed up as "precise and efficient". Although traditional breeding and mutagenesis methods have also played a role, they often seem inadequate when dealing with complex traits. In contrast, CRISPR's multiple editing, base modification, and even guided advanced editing methods enable researchers to deal with the issues of gene function and trait improvement more directly (Huang and Lin, 2024). Its simple operation and clear goals have provided new entry points for many old problems in plant biotechnology. In fact, CRISPR/Cas9 initially existed in bacteria merely as an "immune tool" to recognize invading exogenous DNA. It was only later that people gradually realized that customizable Sgrnas could guide Cas proteins to specific gene loci, thereby achieving knockout, insertion or minor base modifications. Due to its relatively simple design and considerable efficiency, it has rapidly become one of the mainstream technologies in plant genetic engineering (Hu et al., 2025). In addition, some improved versions of Cas9 and strategies that support simultaneous editing of multiple genes have further expanded the application scope of this tool. When it comes to specific examples, tomatoes are an unavoidable object. It is not only an important economic crop but also a commonly used model material for studying fruit development due to its relatively complete genomic data and mature transformation system (Reem and Van Eck, 2019). However, the genetic diversity of tomatoes is relatively limited, which instead prompts researchers to use CRISPR to improve fruit quality and enhance traits such as disease resistance and stress tolerance (Saikia et al., 2024). By rapidly manufacturing targeted mutants, the breeding cycle can be significantly shortened, and the related functional gene analysis can also be advanced more quickly. This study does not focus on the editing principle of CRISPR itself, but rather on a more easily overlooked yet extremely crucial step, the design of gRNA. Good gRNA can enhance the success rate of editing, reduce
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