A mechanistic study on the tolerance of PAM distal end mismatch by SpCas9.

Autor:	Dey D; Department of Natural Products, National Institute of Pharmaceutical Education and Research, Kolkata, West Bengal, India., Chakravarti R; Department of Natural Products, National Institute of Pharmaceutical Education and Research, Kolkata, West Bengal, India., Bhattacharjee O; Plant-Microbe Interaction Division, National Institute of Plant Genome Research, Delhi, India., Majumder S; Department of Life Sciences, Presidency University, Kolkata, India., Chaudhuri D; Department of Life Sciences, Presidency University, Kolkata, India., Ahmed KT; Department of Natural Products, National Institute of Pharmaceutical Education and Research, Kolkata, West Bengal, India., Roy D; Department of Natural Products, National Institute of Pharmaceutical Education and Research, Kolkata, West Bengal, India., Bhattacharya B; Department of Natural Products, National Institute of Pharmaceutical Education and Research, Kolkata, West Bengal, India., Arya M; Department of Natural Products, National Institute of Pharmaceutical Education and Research, Kolkata, West Bengal, India., Gautam A; Algorithms in Bioinformatics, Institute for Bioinformatics and Medical Informatics, University of Tübingen, Tübingen, Baden-Württemberg, Germany; International Max Planck Research School 'From Molecules to Organisms', Max Planck Institute for Biology, Tübingen, Baden-Württemberg, Germany., Singh R; Department of Natural Products, National Institute of Pharmaceutical Education and Research, Kolkata, West Bengal, India., Gupta R; Infectious Diseases and Immunology Division, Indian Institute of Chemical Biology, Kolkata, West Bengal, India., Ravichandiran V; Department of Natural Products, National Institute of Pharmaceutical Education and Research, Kolkata, West Bengal, India., Chattopadhyay D; Sister Nivedita University, Kolkata, West Bengal, India., Ghosh A; Department of Biochemistry, Bose Institute, Kolkata, West Bengal, India., Giri K; Department of Life Sciences, Presidency University, Kolkata, India., Roy S; Infectious Diseases and Immunology Division, Indian Institute of Chemical Biology, Kolkata, West Bengal, India., Ghosh D; Department of Natural Products, National Institute of Pharmaceutical Education and Research, Kolkata, West Bengal, India. Electronic address: dipanjan4u@gmail.com.
Jazyk:	angličtina
Zdroj:	The Journal of biological chemistry [J Biol Chem] 2024 Jul; Vol. 300 (7), pp. 107439. Date of Electronic Publication: 2024 Jun 03.
DOI:	10.1016/j.jbc.2024.107439
Abstrakt:	The therapeutic application of CRISPR-Cas9 is limited due to its off-target activity. To have a better understanding of this off-target effect, we focused on its mismatch-prone PAM distal end. The off-target activity of SpCas9 depends directly on the nature of mismatches, which in turn results in deviation of the active site of SpCas9 due to structural instability in the RNA-DNA duplex strand. In order to test the hypothesis, we designed an array of mismatched target sites at the PAM distal end and performed in vitro and cell line-based experiments, which showed a strong correlation for Cas9 activity. We found that target sites having multiple mismatches in the 18th to 15th position upstream of the PAM showed no to little activity. For further mechanistic validation, Molecular Dynamics simulations were performed, which revealed that certain mismatches showed elevated root mean square deviation values that can be attributed to conformational instability within the RNA-DNA duplex. Therefore, for successful prediction of the off-target effect of SpCas9, along with complementation-derived energy, the RNA-DNA duplex stability should be taken into account. Competing Interests: Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)
Databáze:	MEDLINE
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