CRISPR/Cas9 Driven Targeted Editing of Grain Number 1a gene: sgRNA Constructs Design for Yield Enhancement in Rice (Oryza sativa L.)

Abstract

Yield is the ultimate trait determined by various quantitative trait loci (QTLs). One such QTL, GRAIN NUMBER 1a (GN1a), encodes for an enzyme Cytokinin oxidase/ dehydrogenase (CKX), which negatively influences the yield by degrading the phytohormone Cytokinin. CRISPR/Cas9, a precise means for targeted editing of a gene for the improvement of a particular trait in plants. Therefore, targeted genome editing of GN1a gene was performed to down-regulate the expression using CRISPR/Cas9 for the increase in grain number and yield with improved Cytokinin content in the panicle meristem. For the site-targeted mutagenesis, the single guide RNAs (sgRNAs) were designed using plant specific CRISPR-P v2.0 software. Two efficient sgRNAs were chosen critically entrenched on their GC content, on-target values, location on the gene, off-target sites and their location, secondary structures, adjacent to the Protospacer Adjacent Motif (PAM) NGG. The binary vector pRGEB32, with Cas9 influenced by rice ubiquitin promoter and BsaI restriction site driven by rice U3 promoter was employed for cloning of sgRNAs. The sgRNAs were tempered, phosphorylated and astringed with the binary vector pRGEB32, transformed into E. coli DH5α initially, then mobilized into A. tumefaciens EHA105. The present study helps in the development of elite lines which will lead to enhancement of grain number and overall yield for the growing population and farmer’s welfare.