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A Targeted and Tuneable DNA Damage Tool Using CRISPR/Cas9

Emmanouilidis, Ioannis, Fili, Natalia, Cook, Alexander W., Hari-Gupta, Yukti, dos Santos, Ália, Wang, Lin, Martin-Fernandez, Marisa L., Ellis, Peter J.I., Toseland, Christopher P. (2021) A Targeted and Tuneable DNA Damage Tool Using CRISPR/Cas9. Biomolecules, 11 (2). Article Number 288. E-ISSN 2218-273X. (doi:10.3390/biom11020288) (KAR id:86642)

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Official URL
https://doi.org/10.3390/biom11020288

Abstract

Mammalian cells are constantly subjected to a variety of DNA damaging events that lead to the activation of DNA repair pathways. Understanding the molecular mechanisms of the DNA damage response allows the development of therapeutics which target elements of these pathways. Double-strand breaks (DSB) are particularly deleterious to cell viability and genome stability. Typically, DSB repair is studied using DNA damaging agents such as ionising irradiation or genotoxic drugs. These induce random lesions at non-predictive genome sites, where damage dosage is difficult to control. Such interventions are unsuitable for studying how different DNA damage recognition and repair pathways are invoked at specific DSB sites in relation to the local chromatin state. The RNA-guided Cas9 (CRISPR-associated protein 9) endonuclease enzyme is a powerful tool to mediate targeted genome alterations. Cas9-based genomic intervention is attained through DSB formation in the genomic area of interest. Here, we have harnessed the power to induce DSBs at defined quantities and locations across the human genome, using custom-designed promiscuous guide RNAs, based on in silico predictions. This was achieved using electroporation of recombinant Cas9-guide complex, which provides a generic, low-cost and rapid methodology for inducing controlled DNA damage in cell culture models. View Full-Text

Item Type: Article
DOI/Identification number: 10.3390/biom11020288
Subjects: Q Science > QH Natural history > QH301 Biology
Q Science > QH Natural history > QH426 Genetics
Q Science > QP Physiology (Living systems) > QP506 Molecular biology
Q Science > QP Physiology (Living systems) > QP517 Biochemistry
Divisions: Divisions > Division of Natural Sciences > School of Biosciences
Depositing User: Peter Ellis
Date Deposited: 17 Feb 2021 15:56 UTC
Last Modified: 18 Feb 2021 10:10 UTC
Resource URI: https://kar.kent.ac.uk/id/eprint/86642 (The current URI for this page, for reference purposes)
Ellis, Peter J.I.: https://orcid.org/0000-0001-9709-7934
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