期刊
CELL REPORTS
卷 14, 期 6, 页码 1555-1566出版社
CELL PRESS
DOI: 10.1016/j.celrep.2016.01.019
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资金
- National Cancer Institute (NCI) Cancer Center Support [P30CA16672]
- Cancer Prevention Research Institute of Texas [R1204]
- Odyssey Program at The University of Texas MD Anderson Cancer Center
- Research Fellowship of the Deutsche Forschungsgemeinschaft (DFG)
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 wildtype 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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