Computational Design and Screening of Optimal CRISPR/Cas9 gRNAs Targeting Anthocyanidin synthase (ANS) in Crops
Prachi Digpaul
Department of Microbiology, Choudhary Charan Singh University, Meerut – 250 004, Uttar Pradesh, India.
Ramakrishnaiah Thippenehalli Narasimaiah
Department of Biotechnology and Genetics, M.S. Ramaiah College of Arts, Science and Commerce (Autonomous), 7th Main Rd, MSRIT, M S R Nagar, Mathikere, Bengaluru - 560 054, Karnataka, India.
Krishnagowdu Saravanan
*
Post Graduate and Research Centre in Biotechnology, School of Life Sciences, Arignar Anna College (Arts & Science), Krishnagiri – 635 115, (Affiliated to Periyar University, Salem – 636 011), Tamil Nadu, India.
Lakshmanan Archana Devi
Post Graduate and Research Department of Zoology, Thiagarajar College (Autonomous), Madurai – 625 009, Tamil Nadu, India.
Subbiah Selvarani
Post Graduate and Research Department of Zoology, Thiagarajar College (Autonomous), Madurai – 625 009, Tamil Nadu, India.
Sundarasamy Dhanapal
Post Graduate and Research Centre in Biotechnology, School of Life Sciences, Arignar Anna College (Arts & Science), Krishnagiri – 635 115, (Affiliated to Periyar University, Salem – 636 011), Tamil Nadu, India.
Chinnappan Deepa Joan of Arc
Post Graduate and Research Centre in Biotechnology, School of Life Sciences, Arignar Anna College (Arts & Science), Krishnagiri – 635 115, (Affiliated to Periyar University, Salem – 636 011), Tamil Nadu, India.
*Author to whom correspondence should be addressed.
Abstract
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.
Keywords: Anthocyanidin synthase (ANS), CRISPR/Cas9, guide RNA, CHOPCHOP, CRISPOR, flavonol biosynthesis