Computational Design and Screening of Optimal CRISPR/Cas9 gRNAs Targeting Anthocyanidin synthase (ANS) in Crops

Flavonols are an important class of flavonoids that play a role in plant growth, development, and environmental stress mitigation and have well-documented health-promoting activities. Targeted manipulation of flavonoid biosynthesis has emerged as a promising approach for improving crop nutritional quality through precision genome editing. Anthocyanidin synthase (ANS) catalyses the final step in anthocyanin biosynthesis and represents an important point for redirecting metabolic flux within the flavonoid biosynthetic pathway. In this study, an in silico approach was used to identify and assess high-confidence CRISPR/Cas9 guide RNAs (gRNAs) targeting the ANS in selected cereal crops (Oryza sativa and Zea mays). The ANS sequences of Oryza sativa and Zea mays were retrieved from the NCBI GenBank database and subjected to guide RNA prediction using the CHOPCHOP platform. Candidate gRNAs were subsequently evaluated using CRISPOR based on their Massachusetts Institute of Technology (MIT) specificity score, Cutting Frequency Determination (CFD) score, and predicted off-target profiles. Twelve candidate gRNAs were identified for each of rice and maize. Based on the CRISPOR-based specificity assessment, OsANS-5 and OsANS-8 were identified as the most suitable guide RNAs for O. sativa, each with MIT specificity and CFD scores of 100 and three predicted off-target sites. In Z. mays, ZmANS-9 was identified as the most suitable candidate, with an MIT specificity score of 99, a CFD score of 100, and six predicted off-target sites. Comparative analysis indicated that combining CHOPCHOP for guide RNA design with CRISPOR for specificity assessment helped distinguish guide RNAs with high predicted specificity from candidates with greater predicted off-target potential. These results underscore the value of comprehensive computational screening before experimental genome editing in crops. The guide RNAs identified in this study provide candidates for future CRISPR/Cas9-mediated disruption of the ANS to explore its role in the flavonoid biosynthesis pathway. This workflow also provides a computational framework for functional genomics and future metabolic engineering studies focused on understanding flavonoid pathway regulation and evaluating the potential effects of ANS knockout on flavonol accumulation in crop plants.