Understanding the expression stage of CRISPR-Cas defense system in Leptospira interrogans

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dc.contributor.authorPrakash, Aman
dc.date.accessioned2024-12-10T11:27:07Z
dc.date.available2024-12-10T11:27:07Z
dc.date.issued2023
dc.descriptionSupervisor: Kumar, Manish
dc.description.abstractThe pathophysiology of Leptospirosis, caused by pathogenic Leptospira spp., is unknown mainly due to the lack of efficient genetic manipulation tools. Thus, harnessing the endogenous CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated proteins) system of Leptospira is an attractive strategy to study its pathogenesis; however, it relies on the understanding of the CRISPR-Cas immunity process. This study characterizes the CRISPR arrays and CRISPR-associated proteins (LinCas6, LinCas5, and LinCas3) involved in the expression and interference of RNA-mediated immunity. In L. interrogans, we account for the transcriptionally active CRISPR arrays in the direction of cas-operons. The recombinant LinCas6 (rLinCas6) overexpressed and purified in this study can process the precursor-CRISPR RNA (pre-crRNA) to generate mature crRNA and remains bound with it. The rLinCas6 follows single turnover kinetics where substituting one of the predicted active site residues (His38) reduced cleavage activity on its cognate repeat RNA. Biochemical analysis of the overexpressed and purified rLinCas5 suggested that it is catalytically inactive on nucleic acids. However, rLinCas5 binds to the rLinCas6-crRNA complex essential for stabilizing the mature crRNA during interference. Similarly, the overexpressed and purified rLinCas3 nuclease activity demonstrated that it is a metaldependent nuclease. This study features insight into CRISPR transcription, crRNA biogenesis, and the onset of the effector complex formation in Leptospira, which is essential for RNA-mediated interference of invading nucleic acids. In addition, this study proposes the physiological requirements of Leptospira CRISPR-Cas I-B during interference.
dc.identifier.otherROLL NO.156106021
dc.identifier.urihttps://gyan.iitg.ac.in/handle/123456789/2718
dc.language.isoen
dc.relation.ispartofseriesTH-3054
dc.titleUnderstanding the expression stage of CRISPR-Cas defense system in Leptospira interrogans
dc.typeThesis
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