Development and assessment of CRISP/Cas9- construct towards drought tolerance in rice (Oryza sativa subsp. indica)

• Main Authors: Samsulrizal, Nurul Hidayah, Md Yusof, Anis Afuza, Mohd Zim, Nurul Asyikin, Tamizi, Amin Asyraf, Salleh, Mohd Syahmi, Ahmad Azmi, Nur Sabrina, Zainuddin, Zarina
• Format: Proceeding Paper
• Language: English
• Published: 2023
• Subjects: S Agriculture (General)
• Online Access: http://irep.iium.edu.my/107920/1/107920_Development%20and%20assessment%20of%20CRISPCas9.pdf; http://irep.iium.edu.my/107920/
• Institution: Universiti Islam Antarabangsa Malaysia

The great majority of the population on earth consume rice (Oryza sativa) as their primary source of carbohydrates, however, seriously threatened by the onset of climate change has posed a significant threat to the cultivation of this staple crop. The revolution of genome editing technology has opened a new era in crop improvement. Hence, this study aims to isolate, characterise, and develop the CRISPR/Cas9 construct for SUMO E2-Conjugating Enzyme (OsSCE1) gene. Development of CRISPR/Cas9 constructs involved several stages, (1) characterisation of OsSCE1 gene, (2) sgRNA design, (3) vector construction, (4) Agrobacterium and in-planta transformation and (5) molecular validations. The OsSCE1 gene was verified by sequencing, showing 99% identity with O. sativa on chromosome 10. The 20 bp sgRNA was designed manually with the help of gRNA prediction programmes including CCTop, and Benchling, and the CRISPR constructs (Level 0, 1 and 2) were confirmed by sequencing. Then, the transgenic rice plants harboring sgRNA: Cas9 targeting IR64 's OsSCE1 gene were generated via Agrobacterium mediated in-planta transformation. Leaves of two-month-old plants were subjected to selection assay (300 mg/L kanamycin) and kanamycin-resistant plants were labelled as putative transformants. Polymerase chain reaction (PCR) amplification of Cas9 and OsSCE1 genes using specific primers verified the integration of the transgenes in 29 T0 lines. The mutations were discovered by examining the targeting location on the genome of the relevant transgenic plants. Sequencing analysis revealed the presence of six transgenic lines (D8, C4, C14, B15, C21, and B18) that exhibited a mutagenesis efficiency of approximately 9% for the OsSCE1 gene. This finding suggests that sgRNA: Cas9-induced gene- targeting could be employed to specifically alter agricultural traits and the CRISPR-Cas9 system has the potential to be a highly effective tool for crop breeding trait enhancements.