Publications of NIPGR Scientists
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Item Revisiting rice transformation for a fail-safe protocol and its application for various gene functional and molecular studies(Springer Nature Publishing AG, 2025) Manna, Mrinalini; Rengasamy, Balakrishnan; Reddy, Malireddy K.; Sinha, Alok KrishnaAn efcient transformation and regeneration system is prerequisite for gene functional studies in rice, the staple food crop of the Asian subcontinent. Despite the availability of a large number of rice transformation protocols, it is hard to fnd a simple and minimum input based, but fail-safe protocol that ensures zero number of escapes (or the non-transformants). The fear of obtaining an unpredictable percentage of escapes at the end of rice tissue culture prompts carrying out multiple batches of transformation which not only consumes costly resources and time but also burdens one with screening of a large number of tissue culture derived plants. In the present study, we have described a simple but fail-safe rice transformation protocol for functional validation of the genes by overexpression and CRISPR-Cas9 mediated gene knockout systems. By taking the advantage of high transformability of japonica rice tissues, we have also revealed that the present rice transformation protocol can be efectively employed to visualize protein localization in various subcellular compartments of rice root and callus tissues, an alternative to conventional tobacco/onion peel infltration or protoplast transformation which are either time consuming or tricky. Additionally, the paper also discusses the importance of Southern blotting in gene overexpression studies, utility of non-conventional antibiotic selection approaches and signifcance of sgRNA designing for gene knockout studies in rice. Various troubleshooting advice are also being presented. Overall, the present protocol might serve as an excellent guide for functional validation of several genes present in the rice genome waiting to be explored.Item Breeding rice for yield improvement through CRISPR/Cas9 genome editing method: current technologies and examples(Springer Nature Publishing AG, 2024) Rengasamy, Balakrishnan; Manna, Mrinalini; Thajuddin, Nargis Begum; Sathiyabama, Muthukrishnan; Sinha, Alok KrishnaThe impending climate change is threatening the rice productivity of the Asian subcontinent as instances of crop failures due to adverse abiotic and biotic stress factors are becoming common occurrences. CRISPR-Cas9 mediated genome editing ofers a potential solution for improving rice yield as well as its stress adaptation. This technology allows modifcation of plant’s genetic elements and is not dependent on foreign DNA/gene insertion for incorporating a particular trait. In this review, we have discussed various CRISPR-Cas9 mediated genome editing tools for gene knockout, gene knock-in, simultaneously disrupting multiple genes by multiplexing, base editing and prime editing the genes. The review here also presents how these genome editing technologies have been employed to improve rice productivity by directly targeting the yield related genes or by indirectly manipulating various abiotic and biotic stress responsive genes. Lately, many countries treat genome-edited crops as non-GMOs because of the absence of foreign DNA in the fnal product. Thus, genome edited rice plants with improved yield attributes and stress resilience are expected to be accepted by the public and solve food crisis of a major portion of the globe.Item A simplified and improved protocol of rice transformation to cater wide range of rice cultivars(Springer Nature Publishing AG, 2024) Rengasamy, Balakrishnan; Manna, Mrinalini; Jonwal, Sarvesh; Sathiyabama, Muthukrishnan; Thajuddin, Nargis Begum; Sinha, Alok KrishnaThe latest CRISPR-Cas9-mediated genome editing technology is expected to bring about revolution in rice yield and quality improvement, and thus validation of rice transformation protocols using CRISPR-Cas9-gRNA constructs is the need of the hour. Moreover, regeneration of more number of transgenic rice plants is prerequisite for developing genome-edited rice lines, as recalcitrant rice varieties were shown to have lower editing efciencies which necessities screening of large number of transgenic plants to fnd the suitable edits. In the present study, we have simplifed the Agrobacterium-mediated rice transformation protocol for both Indica and Japonica rice cultivars using CRISPR/Cas9 empty vector construct, and the protocols have been suitably optimized for getting large numbers of the regenerated plantlets within the shortest possible time. The Japonica transgenic lines were obtained within 65 days and for the Indica cultivars, it took about 76–78 days. We also obtained about 90% regeneration efciency for both Japonica and Indica cultivars. The transformation efciency was about 97% in the case of Japonica and 69–83% in the case of Indica rice cultivars. Furthermore, we screened the OsWRKY24 gene editing efciency by transforming rice cultivars with CRISPR/Cas9 construct harbouring sgRNA against OsWRKY24 gene and found about 90% editing efciency in Japonica rice cultivars, while 30% of the transformed Indica cultivars were found to be edited. This implicated the presence of a robust repair mechanism in the Indica rice cultivars.Item Negative regulators of grain yield and mineral contents in rice: potential targets for CRISPR-Cas9-mediated genome editing(Springer Nature Publishing AG, 2023) Yadav, Banita; Majhi, Ashis; Phagna, Kanika; Meena, Mukesh Kumar; Ram, HasthiRice is a major global staple food crop, and improving its grain yield and nutritional quality has been a major thrust research area since last decades. Yield and nutritional quality are complex traits which are controlled by multiple signaling pathways. Sincere efforts during past decades of research have identified several key genetic and molecular regulators that governed these complex traits. The advent of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-mediated gene knockout approaches has accelerated the development of improved varieties; however, finding out target gene with negative regulatory function in particular trait without giving any pleiotropic effect remains a challenge. Here, we have reviewed past and recent literature and identified important negative regulators of grain yield and mineral contents which could be potential targets for CRISPR-Cas9-mediated gene knockout. Additionally, we have also compiled a list of microRNAs (miRNAs), which target positive regulators of grain yield, plant stress tolerance, and grain mineral contents. Knocking out these miRNAs could help to increase expression of such positive regulators and thus improve the plant trait. The knowledge presented in this review would help to further accelerate the CRISPR-Cas9-mediated trait improvement in rice.Item CRISPR-Cas9 system: A new-fangled dawn in gene editing(Elsevier B.V., 2019) Gupta, Darshana; Bhattacharjee, Oindrila; Mandal, Drishti; Sen, Madhab Kumar; Dey, Dhritiman; Dasgupta, Adhiraj; Kazi, Tawsif Ahmed; Gupta, Rahul; Sinharoy, Senjuti; Acharya, Krishnendu; Chattopadhyay, Dhrubajyoti; Ravichandiran, V.; Roy, Syamal; Ghosh, DipanjanTill date, only three techniques namely Zinc Finger Nuclease (ZFN), Transcription-Activator Like Effector Nucleases (TALEN) and Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-Associated 9 (CRISPR-Cas9) are available for targeted genome editing. CRISPR-Cas system is very efficient, fast, easy and cheap technique for achieving knock-out gene in the cell. CRISPR-Cas9 system refurbishes the targeted genome editing approach into a more expedient and competent way, thus facilitating proficient genome editing through embattled double-strand breaks in approximately any organism and cell type. The off-target effects of CRISPR Cas system has been circumnavigated by using paired nickases. Moreover, CRISPR-Cas9 has been used effectively for numerous purposes, like knock-out of a gene, regulation of endogenous gene expression, live-cell labelling of chromosomal loci, edition of single-stranded RNA and high-throughput gene screening. The execution of the CRISPR-Cas9 system has amplified the number of accessible scientific substitutes for studying gene function, thus enabling generation of CRISPR-based disease models. Even though many mechanistic questions are left behind to be answered and the system is not yet fool-proof i.e., a number of challenges are yet to be addressed, the employment of CRISPR-Cas9–based genome engineering technologies will increase our understanding to disease processes and their treatment in the near future. In this review we have discussed the history of CRISPR-Cas9, its mechanism for genome editing and its application in animal, plant and protozoan parasites. Additionally, the pros and cons of CRISPR-Cas9 and its potential in therapeutic application have also been detailed here.Item CRISPR-Cas9 based plant genome editing: Significance, opportunities and recent advances(Elsevier B.V., 2018) Soda, Neelam; Verma, Lokesh; Giri, JitenderPrecise genome editing is a quantum leap in the field of plant sciences. Clustered regularly interspaced short palindromic repeats (CRISPR) and its associated Cas9 protein have emerged as a powerful tool for precise genome editing. CRISPR-Cas9 system introduces small heritable mutations (indels) in the genome of an organism. This system also enables precise gene characterization in plants with complex genomes. Besides, it offers new opportunities of trait stacking, where addition of desirable traits or removal of undesirable traits can be achieved simultaneously in a single event. With CRISPR-Cas9 RNPs technology, raising transgene free genetically modified plants is within realm of possibility which would be helpful in addressing regulatory concerns of transgenic plants. Several new advancements have been made in this technology which has extended its applications in almost every aspect of plant science. For example, recently developed catalytically inactive dCas9 fused with transcriptional effector domains allows targeted activation or silencing of the gene of interest. Apart from this, dCas9 fused with fluorescent labels is a budding tool in chromatin imaging studies. In this review, we summarize these recent advancements in CRISPR/Cas system and methods for analyzing the induced mutations, and its implementations in crop improvement.Item Functional genomics of abiotic stress tolerance in plants: A CRISPR approach(Frontiers Media S.A., 2015) Jain, MukeshVarious abiotic stresses, such as drought, salinity, heat, flooding, ion toxicity and radiation are the major constraints to agricultural production. The understanding of molecular basis of plant response to these environmental conditions has been a major focus of research in the past decades. Several genes/pathways and regulatory networks involved in stress responses have been worked out employing various approaches. Quite a few of these components have been used for engineering abiotic stress tolerance in model and crop plants via classical biotechnological and/or breeding approaches. Success to generate stress-tolerant plants has been achieved to some extent, which has resulted in increased crop yield (Mickelbart et al., 2015). However, novel strategies are desirable to overcome the limitations of classical methods, such as lack of precision and requirement of substantial time to increase the crop production in the current climate change and ever increasing population scenario. The recent availability of genome editing tools provides ample opportunity to introduce targeted modifications in the genome efficiently to study the functional aspects of various components of the genome in diverse plants and offers potential avenues for production of abiotic stress-tolerant crop plants.