Abstract
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.
Original language | English (US) |
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Pages (from-to) | 1555-1566 |
Number of pages | 12 |
Journal | Cell Reports |
Volume | 14 |
Issue number | 6 |
DOIs | |
State | Published - Feb 16 2016 |
Keywords
- CRISPR
- Cell cycle
- Genome editing
- Homologous recombination
- MALAT1
- Proteolysis
- Synthetic biology
ASJC Scopus subject areas
- General Biochemistry, Genetics and Molecular Biology
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