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.Item Targeting silencing of Root architecture associated 1 gene in rice using CRISPR/Cas9 system(Department of Molecular Biology and Biotechnology, Center for Plant Biotechnology and Molecular Biology, Vellanikkara, 2024-12-12) Arpitha Acharya; Abida, P.SRice (Oryza sativa L.) is the second most cultivated crop, feeding around two-thirds of the population after wheat worldwide (Pirdashti et al., 2009). The semi-aquatic nature of rice makes it more susceptible to water stress (Lafitte et al., 2006). Drought stress is the major obstacle in rice production, affecting around 45% of agricultural areas worldwide (Ambavaram et al., 2014; Todaka et al., 2015; Heinemann et al., 2015). Climate change has resulted in unpredictable, more frequent, and distressing weather patterns, which are likely to continue with increasing global warming (IPCC, 2023). The root structure of a plant plays a significant role in scavenging limited resources and coping with stressed conditions, remains unexplored. Researchers have identified genotypes with deep rooting habits that had a better edge towards growth and survival in stressful conditions before the outbreak of stress period exhibit better productivity (Venuprasad et al., 2002). The ROOT ARCHITECTURE ASSOCIATED 1 (OsRAA1) gene belongs to a new small protein family having GTP binding activity that negatively regulates a wide range of cellular processes and can be controlled by phytohormone signaling, particularly auxin (Ge et al., 2004; Xu et al., 2010). According to Ge et al. (2004), constitutive overexpression of the gene led to an increased number of adventitious roots, reduced gravitropic response, and reduced growth of primary roots in rice. The presence of two auxin response elements (AuRE) suggests that the OsRAA1 gene is probably regulated by auxin (Ge et al., 2004). Xu et al. (2010) found that degradation of RAA1 protein is associated with Anaphase Promoting Complex (APC), ascertaining a functional association between RAA1 and APC/C complex. RAA1 was established as a cell cycle candidate and an APC/C substrate for proteolysis. Degradation of RAA1 by the ubiquitin-proteosome structure is necessary for the transition of the cell cycle to anaphase during root growth in rice (Xu et al., 2010). Although a lot of research conveys the biochemical and regulatory role of OsRAA1, several gaps exist in comprehending the function of the OsRAA1 gene under various stress conditions. Thus, the main goal of this study is to knock out the OsRAA1 gene using CRISPR/Cas9 system to produce mutant rice lines with deeper rooting traits. In the current study, Oryza sativa ssp. japonica cv. Nipponbare was used as plant material. The sequence of OsRAA1 was retrieved from the Rice Annotation Project Database (RAP-DB) and Rice Genome Annotation Project (RGAP). The Locus ID of OsRAA1 was identified from RAP DB. The spacer sequence or guide sequences for the sgRNA that target potential protospacers were designed using the CRISPR-P v2.0 portal. Based on the GC content (60-80%), on-score value, off-target sites, and location in the genome, two 20 bp length gRNAs were selected. The gRNAs located towards the 5’ end of the coding sequence of the gene with fewer off target sites and located mostly on the first or initial exons, were preferred. The secondary structures of the gRNAs were also validated using RNA secondary structure prediction tool. In this study, guide RNA constructs were developed using the pRGEB32 vector. The gRNA scaffold is the vector flanked by BsaI restriction sites, enabling easy insertion of designed spacer sequences. The CRISPR/Cas9 construct for cloning was developed by annealing and ligating the gRNAs to the pRGEB32 vector followed by cloning in E. coli strain DH5α. The putative positive clones were identified by colony and plasmid PCR and further confirmed by Sanger sequencing with M13 reverse primer. The sequences of the clones were confirmed by analyzing the results using BioEdit v7.2 software. The multiple sequence alignment in the software confirmed the presence of both the gRNAs (OsRAA1#R1 and OsRAA1#R2). The vector-gRNA constructs, pRGEB32:OsRAA1#R1 and pRGEB32:OsRAA1#R2 were confirmed positive for cloning after sequence analysis and were further mobilized into Agrobacterium strain EHA105 by freeze-thaw method (Holsters et al., 1978). Randomly selected colonies were screened for inserts by colony PCR with gRNA specific and M13 reverse primer. Expected bands of size ~450 bp were observed on 1% agarose gel. Positive colonies of OsRAA1#R1(4) and OsRAA1#R2(6) constructs in EHA105 were then used for rice genetic transformation. The calli induced from the seeds of rice cv. Nipponbare were inoculated into MS medium supplemented with 2,4-D (2.5 mg/L) for callus induction. After 2 weeks of inoculation, seeds and shoots were removed and only the callus cultures were transferred to fresh callus induction media. The established calli were transformed by co-cultivation with positive EHA colonies. To remove excess Agrobacterium load, calli were washed with a solution of sterile water containing cefotaxime and timentin. The selection of transformed calli was carried out in three steps through selection media I, II, and III for hygromycin resistance. From selection media III, only the micro-calli of the proliferating calli were transferred to regeneration media. In the future, regenerated shoots will be analyzed for mutation and the expected mutant rice lines may confer drought tolerance.Item Editing of rice transcription factor OsMADS26 for drought tolerance through CRISPR/Cas9 system(Department of Plant Biotechnology, College of Horticulture, Vellanikkara, 2021) Anjala, K; Rehna AugustineRice (Oryza sativa L.) is the most widely consumed staple food of world’s human population belonging to Asia and Africa. Being a semi-aquatic annual plant, rice is highly prone to losses due to various environmental stresses. Many studies regarding this had revealed the need for developing varieties tolerant to abiotic and biotic stresses. Various methods like Marker Assisted Breeding, mutation breeding, RNAi, Antisense technology, ZFNs and TALENs were in use to develop elite traits for abiotic stress tolerance in crops like rice. But very recently, CRISPR/Cas9 system had come into the limelight as an efficient tool for the genetic manipulations of crops. Studies have identified OsMADS26 transcription factor as a negative regulator of drought tolerance in rice. Hence the current study, ‘Editing of rice transcription factor OsMADS26 for drought tolerance through CRISPR/Cas9 system’ was undertaken during the period from 2019 to 2021 at the Centre for Plant Biotechnology and Molecular Biology, CoA, Vellanikkara, Thrissur with an objective to develop drought tolerance in rice. The rice cultivar Nipponbare was selected for the study due to its competence in genetic transformation and regeneration. For CRISPR/Cas9 mediated targeted editing of OsMADS26 gene, guide RNAs (gRNAs) were designed using online software CRISPR-P v2. Genome sequence information of OsMADS26 gene available from rice genome annotation project was used for the study. Genomic region of OsMADS26 gene, flanking the gRNA target (~ 450 bp) was amplified using gene specific primers and sequence of the target region was confirmed using BLASTn and ClustalW analysis. The CRISPR/Cas9 binary vector pRGEB32 was used to clone the guide RNAs using BsaI restriction sites. Three gRNAs were selected for cloning based on features like on score value (higher the value better the editing efficiency), GC content, (40-60%), no. of off-target sites (Minimum number of off-target sites preferred), presence of secondary structure, location on the genome (towards 5' end of gene in exonic region is preferred) etc. The CRISPR/Cas9 construct for cloning was developed by annealing and ligating the gRNAs to the pRGEB32 vector followed by cloning in E. coli strain DH5α. The putative positive clones were identified by colony PCR and further confirmed by Sanger sequencing. The plasmids isolated from PCR positive colonies were sequenced using universal M13 Reverse primer which is present on pRGEB32 vector. The sequences of the clones were confirmed using multiple sequence alignment tool ClustalW. One colony of gRNA 1 construct (OsMADS26 #G1-1) and two colonies of gRNA 3 (OsMADS26 #G3-3 and OsMADS26 #G3-4) were found positive. The CRISPR/Cas9 constructs of OsMADS26 were then mobilized into Agrobacterium tumefaciens strain EHA105 following the Freeze-thaw method. The positive clones were identified using plasmid PCR using hygromycin gene specific primers. Positive colonies of OsMADS26 #G1-1 and OsMADS26 #G3-3 constructs in EHA105 were then used for rice genetic transformation. The seeds of Oryza sativa sub species japonica cultivar Nipponbare were inoculated into N6 medium supplemented with 3.0 mgL-1 2,4-D for callus induction. After five days, the calli were infected with Agrobacterium cultures harboring desired gRNA constructs for 1.5-2 min. Along with the gRNA constructs, an empty vector was also transformed to rice as vector control and a set of untransformed culture were also maintained. After around two days of co-cultivation, the excess Agrobacterium growth was washed-off thoroughly from the calli using the bacteriostatic agent Augmentin. The calli were then placed on selection medium containing Augmentin and Hygromycin. The hygromycin resistant calli showed proliferation after 14 days of incubation. The proliferating microcalli were then transferred to regeneration medium after 21 days. Proliferation of microcalli was observed in vector control, wild type as well as OsMADS26 #G1-1 and OsMADS26 #G3-3 co-transformed plates. The vector control and untransformed calli showed greening and shoot primordia initiation in regeneration medium. The regenerated shoots will be analyzed for mutation in future. Hence, in the current study, gRNA constructs for targeted editing of OsMADS26 gene was successfully developed and transformed in to rice cultivar Nipponbare. Rice genetic transformation suitable to our lab conditions were also optimized. Rice plants with mutations in the OsMADS26 gene is expected in future which can confer drought tolerance.