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.
Keywords:
CRISPR; MALAT1; cell cycle; genome editing; homologous recombination; proteolysis; synthetic biology.
Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, Non-P.H.S.
MeSH terms
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Adenomatous Polyposis Coli Protein / genetics
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Adenomatous Polyposis Coli Protein / metabolism
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Antigens, CD
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Bacterial Proteins / genetics*
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Bacterial Proteins / metabolism
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Base Sequence
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CRISPR-Associated Protein 9
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CRISPR-Cas Systems*
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Cadherins / genetics
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Cadherins / metabolism
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DNA / genetics*
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DNA / metabolism
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DNA Breaks, Double-Stranded
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DNA End-Joining Repair*
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Endonucleases / genetics*
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Endonucleases / metabolism
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G1 Phase Cell Cycle Checkpoints / genetics*
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Geminin / genetics
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Geminin / metabolism
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Gene Expression Regulation
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Gene Knock-In Techniques / methods
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Genetic Engineering
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HEK293 Cells
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Humans
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Molecular Sequence Data
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Plasmids / chemistry
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Plasmids / metabolism
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Recombinant Fusion Proteins / genetics
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Recombinant Fusion Proteins / metabolism
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Recombinational DNA Repair*
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Transfection
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Transgenes
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Ubiquitin-Protein Ligases / genetics
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Ubiquitin-Protein Ligases / metabolism
Substances
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APC protein, human
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Adenomatous Polyposis Coli Protein
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Antigens, CD
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Bacterial Proteins
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CDH1 protein, human
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Cadherins
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GMNN protein, human
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Geminin
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Recombinant Fusion Proteins
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DNA
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Ubiquitin-Protein Ligases
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CRISPR-Associated Protein 9
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Cas9 protein, Francisella novicida
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Endonucleases