Post-translational regulation of Cas9 during G1 enhances homology-directed repair
CRISPR/Cas9 induces DNA double-strand breaks that are repaired by cell-autonomous repair pathways, namely, non-homologous end-joining (NHEJ), or homology-directed repair (HDR). While HDR is absent in G1, NHEJ is active throughout the cell cycle and, thus, is largely favored over HDR. We devised a st...
Gespeichert in:
| Hauptverfasser: | , |
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| Dokumenttyp: | Article (Journal) |
| Sprache: | Englisch |
| Veröffentlicht: |
February 4, 2016
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| In: |
Cell reports
Year: 2016, Jahrgang: 14, Heft: 6, Pages: 1555-1566 |
| ISSN: | 2211-1247 |
| DOI: | 10.1016/j.celrep.2016.01.019 |
| Online-Zugang: | Verlag, Volltext: https://doi.org/10.1016/j.celrep.2016.01.019 Verlag: http://www.sciencedirect.com/science/article/pii/S2211124716000401 
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| Verfasserangaben: | Tony Gutschner, Monika Haemmerle, Giannicola Genovese, Giulio F. Draetta, and Lynda Chin |
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| 520 | |a CRISPR/Cas9 induces DNA double-strand breaks that are repaired by cell-autonomous repair pathways, namely, non-homologous end-joining (NHEJ), or homology-directed repair (HDR). While HDR is absent in G1, NHEJ is active throughout the cell cycle and, thus, is largely favored over HDR. We devised a strategy to increase HDR by directly synchronizing the expression of Cas9 with cell-cycle progression. Fusion of Cas9 to the N-terminal region of human Geminin converted this gene-editing protein into a substrate for the E3 ubiquitin ligase complex APC/Cdh1, resulting in a cell-cycle-tailored expression with low levels in G1 but high expression in S/G2/M. Importantly, Cas9-hGem(1/110) increased the rate of HDR by up to 87% compared to wild-type Cas9. Future developments may enable high-resolution expression of genome engineering proteins, which might increase HDR rates further, and may contribute to a better understanding of DNA repair pathways due to spatiotemporal control of DNA damage induction. | ||
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