1. KAUTIR (Kerala Agricultural University Theses Information and Retrieval)
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Item Targeted editing of Grain number 1a gene in rice using CRISPR/Cas9 system(Department of Molecular Biology and Biotechnology, Centre for Plant Biotechnology and Molecular Biology, College of Agriculture, Vellanikkara, 2024-12-27) Bhavya, G; Abida, P SRice (Oryza sativa L.) is one of the important and primary cereal food crop, that provide nutrition for almost half of the world's population. As the global population continues to grow, the demand for rice will rise substantially. It is anticipated that to satisfy the food needs of an expanding population, rice production will have to increase proportionately. Yield is considered to be the most complex and significant physiological trait governed by various external factors like abiotic, biotic, etc and internal factors like genetic, biochemical, etc. Significant depletion in the yield can be caused by physiological, environmental, and morphological hindrances which have a massive impact on the growth and development of plant. Cytokinins (CKs) have a unique role in the growth and development, especially plays a prominent part in the regulation of panicle architecture which determines grain number in rice (Azizi et al., 2015; Yeh et al., 2015). Previous studies shown that yield is influenced by many quantitative trait loci (QTLs). One such QTL, GRAIN NUMBER1a (GN1a), encodes an enzyme Cytokinin oxidase 2/dehydrogenase (OsCKX2) which negatively affects the yield by degradation of Cytokinin in rice (Ashikari et al., 2005). Various genetic engineering techniques like gene silencing methods (Anti-sense technology, RNAi technology), and gene knock-out techniques (ZFN’s, TALEN’s, CRISPR/Cas system) are being used as efficient and precise tools for the development of elite varieties. But, CRISPR/Cas system is most widely used because of its high target specificity, and silencing efficiency for many crops. Therefore, the present study was initiated to knock-out the OsGN1a gene using CRISPR/Cas9 technique with an intention to develop elite lines with yield enhancement in rice. The rice japonica cultivar Nipponbare was used for the study because of its efficient genetic transformation and regeneration ability. Gene sequence information of OsGN1a was downloaded from Rice Genome Annotation Project. For CRISPR/Cas9 mediated site-targeted mutagenesis, single guide RNAs (sgRNAs) were designed using plant specific CRISPR-P v2.0 software. Two best sgRNAs were selected based on their on-target scores, GC content, location on the gene, off-target scores, sites and their location, and secondary structures. The CRISPR/Cas9 binary vector pRGEB32 with BsaI restriction sites was used to clone sgRNAs. The CRISPR/Cas9 cassette for editing was constructed by annealing, phosphorylating and ligating the sgRNAs into the pRGEB32 vector succeeded by transformation into E. coli DH5α strain with heat-shock method @ 42°C for 90 sec (Chang et al., 2017). The transformed clones were identified by colony PCR. The PCR positive colonies (colony 4 of OsGN1a#G1 and colony 1 of OsGN1a#G2) were used for plasmid isolation, plasmid PCR and further confirmed by Sanger sequencing using universal M13 reverse primer. The sequencing results were analyzed using sequence alignment editor BioEdit 7.2 software which confirmed that both sgRNAs inserted into vector backbone. The recombinant CRISPR/Cas9 constructs were then mobilized into A. tumefaciens EHA105 strain through freeze-thaw method @ 37°C for 5 min (Holsters et al., 1978). The transformed clones were identified by colony PCR. The PCR positive colonies (colony 4 of OsGN1a#G1 and colony 6 of OsGN1a#G2) were used for plasmid isolation, and re confirmed by plasmid PCR. Agrobacterium mediated genetic transformation or rice was performed according to Raineri et al. (1990) with minor modifications. The mature dehusked seeds of Oryza sativa ssp. japonica cultivar Nipponbare were surface sterilized and inoculated on MS media supplemented with 2,4-D (2.5 mg/L) for callus induction. After 14 days, the seeds and shoots formed were removed and the calli were sub-cultured on fresh media. The 21-days old calli were agro infected by co-cultivation of calli with A. tumefaciens harboring preferred sgRNA constructs. After two days of co-cultivation, the excess A. tumefaciens load was removed with antibiotics Cefotaxime (250 mg/L) and Timentin (200 mg/L). The blot dried calli were then inoculated on selection media-I, supplemented with Cefotaxime, Timentin and Hygromycin (50 mg/L) for 10 days. The transformed calli initially turned brown but showed proliferation after 10 days of incubation on selection media-II, and then only the proliferated calli was transferred to selection media-III and allowed to proliferate for 7 days. The proliferating micro calli were then transferred to regeneration media supplemented with NAA (0.5 mg/L), BAP (2.5 mg/L), Kinetin (0.5 mg/L) and Hygromycin after 27 days. The calli of both constructs (OsGN1a#G1 and OsGN1a#G2 respectively) displayed greening and shoot primordia on regeneration media. The regenerated shoots will be further analyzed for mutation in future. Hence, in the present study, sgRNA constructs for targeted editing of OsGN1a gene were successfully developed and transformed into rice japonica cultivar Nipponbare. In future, rice plants with mutations in the OsGN1a gene is expected which will lead to the enhancement of grain number and overall yield.