The rice response regulator 22 (RR22) has been reported to be negatively regulating salt tolerance in Oryza sativa and involved in the cytokinin signaling pathway; however, it has not been documented in any Malaysian rice and is deemed as an appealing subject for CRIPSR/Cas9 editing. This study analysed the OsRR22 gene from the Malaysian rice cultivar ‘MR 219’ to elucidate its function and determine the gRNA target site, in which the finding served as the most vital part for our current CRISPR/Cas9 genome editing experiment. In brief, the methods employed include total genome isolation, polymerase chain reaction (PCR) amplification, DNA sequencing, genome search, and computational analyses involving an array of in silico tools. The transcript of OsRR22 was 2,019 bp long, composed of six exons and it encoded a highly conserved 696 amino acid residues. Motif analysis revealed the gene product, RR22, contained response regulator (RR) receiver domain (position 27-142), disordered domain (position 154-214), and Myb-like DNA-binding domain (position 214-273). Analysis on the protein-protein interaction (PPI) using STRING revealed RR22 interacted with various proteins including RR24, RR B8A7T0, and a set of HPt domain-containing proteins (B8B4B1, B8AYV8, B8BEM5, B8A9E0 and B8B9H1). Analysis of gene expression profiles via the Rice Expression Database (RED) revealed the OsRR22 (Os06g0183100) gene was highly expressed (FPKM>10) in the leaf and root. In addition, there were 15 genes to be co-expressed (Pearson’s r value > 0.85) with the OsRR22 gene of which high-affinity potassium transporter 9 (HAK9, Os07g0679000) was one of them. Based on the first exon of OsRR22 that encoded a part of the RR receiver domain, a CRISPR-gRNA 20-bp spacer was generated through CCTop. The gRNA spacer was synthesised, annealed, and ligated into the CRSPR/Cas9 pRGEB32 vector. The CRISPR/Cas9 construct—targeting MR 219’s OsRR22 and intended for Agrobacterium-based delivery—was successfully developed. Our study here documents the upstream workflow involved in rice genome editing with an emphasis on the gene characterisation through multiple bioinformatics tools
