Gravells, P;
Ahrabi, S;
Vangala, RK;
Tomita, K;
Brash, JT;
Brustle, LA;
Chung, C;
... Porter, AC; + view all
(2015)
Use of the HPRT gene to study nuclease-induced DNA double-strand break repair.
Human Molecular Genetics
, 24
(24)
pp. 7097-7110.
10.1093/hmg/ddv409.
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Abstract
Understanding the mechanisms of chromosomal double-strand break repair (DSBR) provides insight into genome instability, oncogenesis and genome engineering, including disease gene correction. Research into DSBR exploits rare-cutting endonucleases to cleave exogenous reporter constructs integrated into the genome. Multiple reporter constructs have been developed to detect various DSBR pathways. Here, using a single endogenous reporter gene, the X-chromosomal disease gene encoding hypoxanthine phosphoribosyltransferase (HPRT), we monitor the relative utilization of three DSBR pathways following cleavage by I-SceI or CRISPR/Cas9 nucleases. For I-SceI, our estimated frequencies of accurate or mutagenic non-homologous end-joining and gene correction by homologous recombination are 4.1, 1.5 and 0.16%, respectively. Unexpectedly, I-SceI and Cas9 induced markedly different DSBR profiles. Also, using an I-SceI-sensitive HPRT minigene, we show that gene correction is more efficient when using long double-stranded DNA than single- or double-stranded oligonucleotides. Finally, using both endogenous HPRT and exogenous reporters, we validate novel cell cycle phase-specific I-SceI derivatives for investigating cell cycle variations in DSBR. The results obtained using these novel approaches provide new insights into template design for gene correction and the relationships between multiple DSBR pathways at a single endogenous disease gene.
| Type: | Article |
|---|---|
| Title: | Use of the HPRT gene to study nuclease-induced DNA double-strand break repair. |
| Location: | England |
| Open access status: | An open access version is available from UCL Discovery |
| DOI: | 10.1093/hmg/ddv409 |
| Publisher version: | http://dx.doi.org/ 10.1093/hmg/ddv409 |
| Language: | English |
| Additional information: | Copyright © The Author 2015. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. |
| Keywords: | Animals, Bacterial Proteins, CRISPR-Cas Systems, Cell Cycle, Cell Line, Tumor, DNA Breaks, Double-Stranded, DNA Repair, Deoxyribonucleases, Type II Site-Specific, Endonucleases, Genes, Reporter, HeLa Cells, Humans, Hypoxanthine Phosphoribosyltransferase, Mice, Mutagenesis, Saccharomyces cerevisiae Proteins |
| 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 Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences > Cancer Institute UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Medical Sciences > Cancer Institute > Research Department of Cancer Bio |
| URI: | https://discovery.ucl.ac.uk/id/eprint/1482974 |
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