T. Tayalkar, A. Rani, A. Jajoo, A. K. Anshu, V. Kumar Soybean, CRISPER/ Cas9, long-juvenile, gRNAs, GmFT2a 70-83 10(2) 2026 April 2026 Coming soon Effort was made to design CRISPER/ Cas9 guide RNA for long-juvenile trait in soybean. The long-juvenile trait is a crucial agronomic characteristic that extends the vegetative growth phase and delays flowering in soybean grown under short-day photoperiod conditions. GmFT2a (Glyma.16g150700) is a major gene responsible for early flowering under short-day conditions in soybean. CRISPR-Cas9-mediated genome editing is the most advanced and rapid process for genome editing, which requires the design and selection of unique guide RNA (gRNA) specific to the targeted gene. For CRISPR-Cas9-mediated editing of the GmFT2a gene, a total of 25 gRNAs targeting the first exon were designed using the Cas-Designer component of CRISPR RGEN tools. For cleavage efficiency analysis, a total of 9 gRNAs were selected based on minimum off-target sites (less than 30) and efficacy scores greater than 0.60. After cleavage efficiency analysis, three gRNAs were found to cleave their respective target sites more efficiently than the rest of the gRNAs. In silico off-target analysis further confirmed the high specificity and precision of the selected gRNAs for targeted editing of the GmFT2a locus. The optimized CRISPR-Cas9 construct developed in this study provides a promising strategy for generating long-juvenile soybean genotypes adaptable to tropical and subtropical agro-climatic conditions. Out of 3 gRNAs, one gRNA was inserted into the CRISPR/Cas9 expression vector “Cas9_MDC123” with the GmU6 promoter and scaffold and cloned into Agrobacterium tumefaciens strain EHA105 for editing of GmFT2a gene for the development of a soybean genotype with long-juvenile trait.