Publications of NIPGR Scientists

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    A CRISPR-Cas9 library to target putative redundant gene sets facilitates their functional exploration in grain development in rice
    (Springer Nature Publishing AG, 2025) Yadav, Banita; Sardar, Shaswati; Yadav, Anil; Kumari, Annapurna; Gautam, Mohini; Mandlik, Rushil; Arora, Simran; Kumar, Shailesh; Jewaria, Pawan Kumar; Sonah, Humira; Deshmukh, Rupesh; Chinnusamy, Viswanathan; Ram, Hasthi
    Advent of CRISPR-Cas9 library approach has revolutionized the field of high throughput targeted mutagenesis in plants. By identifying an sgRNA spacer that can target multiple paralogous genes in a genome, higher-order knockout plants can be developed. Using this concept, we developed ten CRISPR-Cas9 pool libraries and generated higher-order knockout plants in rice. Towards this, firstly we identified genome-wide sets of genes which are co-expressed and have high sequence similarity and can be targeted by a single sgRNA. Based on the expression pattern, these genes were divided into ten groups, and subsequently ten CRISPR-Cas9 plasmid libraries were developed. One such library designed against seed-expressed genes was transformed into rice and higher-order knockout plants were developed. Genotyping revealed that around 90% T0 plants had editing, and among the edited plants majority of them were higher-order knockouts. Phenotypic analysis in the next generation discovered functions of several seed specific genes in grain length, width, number and 100-grain weight. By analyzing single and double mutants for two Agenet domain-containing proteins, we have discovered an epistatic interaction between them for grain development. Further application of our approach will help to uncover hidden functions of the targeted genes and accelerate functional genomics research in rice. The CRISPR-Cas9 library is a useful approach to generate higher-order knockout mutants and identify functions of the targeted genes in rice.
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    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, Hasthi
    Rice 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.
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    Dissecting the nutrient partitioning mechanism in rice grain using spatially resolved gene expression profiling
    (Oxford University Press, 2021) Ram, Hasthi; Singh, Anmol; Katoch, Megha; Kaur, Ravneet; Sardar, Shaswati; Palia, Shubham; Satyam, Rohit; Sonah, Humira; Deshmukh, Rupesh; Pandey, Ajay Kumar; Gupta, Ishaan; Sharma, Tilak Raj
    Rice, a staple food worldwide, contains varying amounts of nutrients in different grain tissues. The underlying molecular mechanism of such distinct nutrient partitioning remains poorly investigated. Here, an optimized rapid laser capture microdissection (LCM) approach was used to individually collect pericarp, aleurone, embryo and endosperm from grains 10 days after fertilization. Subsequent RNA-Seq analysis in these tissues identified 7760 differentially expressed genes. Analysis of promoter sequences of tissue-specific genes identified many known and novel cis-elements important for grain filling and seed development. Using the identified differentially expressed genes, comprehensive spatial gene expression pathways were built for accumulation of starch, proteins, lipids, and iron. The extensive transcriptomic analysis provided novel insights about nutrient partitioning mechanisms; for example, it revealed a gradient in seed storage protein accumulation across the four tissue types analysed. The analysis also revealed that the partitioning of various minerals, such as iron, is most likely regulated through transcriptional control of their transporters. We present the extensive analysis from this study as an interactive online tool that provides a much-needed resource for future functional genomics studies aimed to improve grain quality and seed development.