Publications of NIPGR Scientists

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    Branched DNA processing by a thermostable CAS-Cas4 from Thermococcus onnurineus: expanding biochemical landscape of nuclease activity
    (Elsevier B.V., 2025) Jain, Muskan; Pattnayak, Asish Kumar; Aggarwal, Sakshi; Rai, Praveen; Kavya, J.; Chandrayan, Sanjeev; Goel, Manisha; Gaur, Vineet
    The adaptive immune function of CRISPR-Cas systems in bacteria and archaea is mediated through CRISPR-Associated Proteins (Cas). The adaptation module, typically involving Cas1, Cas2, and Cas4, helps integrate viral “spacer” sequences into the host genome. Cas4 proteins are classified into two types based on neighboring genes: CAS-Cas4, flanked by other cas genes, and Solo-Cas4, which exist independently. While CAS-Cas4 proteins are implicated in adaptation, they remain biochemically uncharacterized in archaea, unlike archaeal Solo-Cas4 proteins. This study biochemically characterizes TON_0321, a CAS-Cas4 protein from the Type IV-C CRISPR cassette of Thermococcus onnurineus. TON_0321 exhibits 5′ to 3′ exonuclease activity and unique structure-dependent endonuclease activity, shedding light on CAS-Cas4 functional diversity. A distinct spatial organization of the catalytic site, angled with the positively charged patch on the protein surface, enables TON_0321 to recognize branching points in DNA substrates. Furthermore, this spatial arrangement facilitates cleavage 2 to 3 nucleotides away from the branch point in the 5′ direction, demonstrating structure-specific endonuclease activity.
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    CRISPR/Cas9 directed editing of lycopene epsilon-cyclase modulates metabolic flux for β-carotene biosynthesis in banana fruit
    (Elsevier B.V., 2020) Kaur, Navneet; Alok, Anshu; Shivani; Kumar, Pankaj; Kaur, Navjot; Awasthi, Praveen; Chaturvedi, Siddhant; Pandey, Pankaj; Pandey, Ashutosh; Pandey, Ajay K.; Tiwari, Siddharth
    Banana is one of the most economically important fruit crops worldwide. Genetic improvement in banana is a challenging task due to its parthenocarpic nature and triploid genome. Genetic modification of crops via the CRISPR/Cas9 module has emerged as a promising tool to develop important traits. In the present work, a CRISPR/Cas9-based approach was used to develop the β-carotene-enriched Cavendish banana cultivar (cv.) Grand Naine (AAA genome). The fifth exon of the lycopene epsilon-cyclase (LCYε) gene was targeted. The targeting specificity of the designed guide-RNA was also tested by its ability to create indels in the LCYε gene at the A genome of cv. Rasthali (AAB genome). Sequence analysis revealed multiple types of indels in the genomic region of Grand Naine LCYε (GN-LCYε). Metabolic profiling of the fruit pulp of selected edited lines showed enhanced accumulation of β-carotene content up to 6-fold (~24 μg/g) compared with the unedited plants. These lines also showed either an absence or a drastic reduction in the levels of lutein and α-carotene, suggesting metabolic reprogramming, without any significant effect on the agro-morphological parameters. In addition, differential expression of carotenoid pathway genes was observed in the edited lines in comparison to unedited plants. Overall, this is the first report in banana to improve nutritional trait by using a precise genome editing approach.