Strategies for developing and evaluating CRISPR/CAS9 construct towards drought tolerance in rice (Oryza sativa ssp. Indica)

• Main Authors: Samsulrizal, Nurul Hidayah, Md Yusof, Anis Afuza, Abdul Sattar, Siti Syafiqa, Mohd Zim, Nurul Asyikin, Salleh, Mohd Syahmi, Zainal, Zamri, Zainuddin, Zarina
• Format: Conference or Workshop Item
• Language: English
• Published: 2022
• Subjects: S Agriculture (General); S494.5.S86 Sustainable agriculture
• Online Access: http://irep.iium.edu.my/98515/1/98515_Strategies%20for%20developing%20and%20evaluating%20crisprcas9.pdf; http://irep.iium.edu.my/98515/
• Institution: Universiti Islam Antarabangsa Malaysia

The majority of the world's population consumes rice (Oryza sativa), which is a staple meal. On the other side, climate change has put rice production in jeopardy. The advent of genome editing technologies has ushered in a new age of agricultural development. Hence, the goal of this work is to use the golden gate cloning approach to produce the CRISPR/Cas9 drought tolerance construct in O. sativa subsp. indica. The development of CRISPR/Cas9 constructs involved several stages, i.e., identification of the SUMO E2-Conjugating Enzyme (OsSCE1) gene, sgRNA selection, and vector construction of the CRISPR/Cas9. The first step in this research was to characterise the OsSCE1 gene using FGENESH, GeneMarkS, InterProScan, and Blast2GO tools. Then, the putative OsSCE1 gene was verified by sequencing, and found to be 99% identical to O. sativa on chromosome 10. The sgRNA design was performed manually with the help of gRNA prediction programmes including WU-CRISPR, CCTop, Benchling, and CRISPR-P. The golden gate cloning method was then used to validate the virtual vector, which included separate vector containing the OsSCE1 sgRNA sequence, planta selectable maker, and Cas9 cassette that were standardised elements for hierarchical modular cloning (MoClo). This study demonstrated that our system, such inclusions are achievable, resulting in a speedier and more efficient assembly process which could contribute to enhancing the resistance and tolerance of rice. To assure a satisfactory outcome, the effectiveness of knocking out candidate genes in O. sativa on drought tolerance will be explored further during the in vivo stage.