Improving rice (Oryza sativa L.) quality is crucial to obtaining local rice with better genetic potential and superiority. The research aimed to construct a CRISPR/Cas9 module cassette and introduce the construct into rice to develop a new non-transgenic superior Mentik Susu variety with early maturity, short stem, high yield, and resistance to bacterial leaf blight. The annealed oligonucleotides of gRNA spacers of the HD2 gene ligated into pDIRECT-21A vector plasmid used the golden gate reaction to construct a CRISPR/Cas9 module cassette. The recombinant plasmid’s verification by digestion engaged a combination of KpnI-HindIII restriction enzymes and Sanger DNA sequencing. The Agrobacterium-mediated co-transformation procedure introduced the CRISPR/Cas9 cassettes (four module cassettes with different gRNAs of the genes, i.e., GA20ox-2, OsCKX2, OsSWEET11, and HD2) into the rice genome with immature rice embryos as explants. Molecular analysis of the transformed T0 putative lines ensued to identify the insertion of T-DNA fragments (containing the Cas9 and hptII genes) and the occurrence of mutagenesis employing PCR amplification and DNA sequencing. The result showed that the CRISPR/Cas9 cassette vector containing the Cas9 gene and the targeted gRNA construction succeeded. Rice transformations of Mentik Susu have generated 157 T0 putative lines, with several of the lines harboring the Cas9 and hptII genes detected positively. Sanger DNA sequencing analysis demonstrated that eight rice lines had a mutation occurrence in the target genes, i.e., two mutations in the OsGA20ox line, five mutations in the OsSWEET11 lines, and one mutation in the Gn-1a (OsCKX2) line. Based on these results, it is probable that the mutant lines also have a phenotype change that is beneficial to produce promising rice genotypes with early maturing, short stems, high yield, and bacterial leaf blight resistance.

Keywords: Oryza sativa L., Mentik Susu rice cultivar, genome editing, CRISPR/Cas9, mutagenesis, early maturity, high yield

Key findings: The presented research has successfully constructed the CRISPR/Cas9-gRNA-HD2 vector cassette and introduced the construct into rice cv. ‘Mentik Susu’ to edit the HD2 gene for developing an early-maturity rice cultivar. Genome editing of the multiple-gene targets (GA20ox-2, OsCKX2, OsSWEET11, and HD2 gene) in rice cv. ‘Mentik Susu’ has resulted in obtaining several rice lines with the CRISPR mutated genes.