Rice (Oryza sativa L.) production on acidic drylands needs optimization through breeding to develop drought-resistant cultivars. The rice cultivar Inpago-5 showed moderate tolerance to drought stress. However, further enhancing this tolerance can proceed by precise gene editing of the DREB (dehydration-responsive element binding) gene, utilizing the CRISPR (clustered regularly interspaced palindromic repeats) /Cas9 system. The presented study aimed to engineer the single-guided RNA (sgRNA) targeting the DREB gene in rice cultivar Inpago-5 using the CRISPR/Cas9 approach. The use of the pRGEB32 plasmid harboring the Cas9 gene helped facilitate the cloning of sgRNA-DREB through the Golden Gate cloning technique and heat shock transformation. The sgRNA-DREB confirmation succeeded through colony PCR (polymerase chain reaction), followed by plasmid isolation and subsequent validation using PCR with specific primers and sequencing primers targeting the pRGEB32 vector. The latest analysis revealed the pRGEB32-sgDREB_sg1 and pRGEB32-sgDREB_sg2 transformed bacteria proliferated on LB+Kanamycin selection media, indicating successful insertion of sgRNA-DREB into the pRGEB32 plasmid. The verification through colony and plasmid PCR with specific primers confirmed the presence of a 425-bp amplicon. Sequencing data showed the correct insertion of sgRNA-DREB sequences at the designated site.

Keywords: Rice (O. sativa L.), cultivar Inpago-5, plant breeding, CRISPR/Cas9, gene editing

Key findings: The gene encoding rice (O. sativa L.) drought tolerance DREB4 succeeded integration into the pRGEB32 vector, with the sgRNA-DREB accurately inserted at the targeted site.