Emmanouilidis, I;
Fili, N;
Cook, AW;
Hari-Gupta, Y;
dos Santos, A;
Wang, L;
Martin-Fernandez, ML;
... Toseland, CP; + view all
(2021)
A Targeted and Tuneable DNA Damage Tool Using CRISPR/Cas9.
Biomolecules
, 11
(2)
, Article 288. 10.3390/biom11020288.
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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.
| Type: | Article |
|---|---|
| Title: | A Targeted and Tuneable DNA Damage Tool Using CRISPR/Cas9 |
| Open access status: | An open access version is available from UCL Discovery |
| DOI: | 10.3390/biom11020288 |
| Publisher version: | https://doi.org/10.3390/biom11020288 |
| Language: | English |
| Additional information: | This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
| Keywords: | DNA damage; Cas9; double-strand break; DNA repair |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Lab for Molecular Cell Bio MRC-UCL |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10135826 |
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