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Cell Rep. 2016 Feb 16;14(6):1555-1566. doi: 10.1016/j.celrep.2016.01.019. Epub 2016 Feb 4.

Post-translational Regulation of Cas9 during G1 Enhances Homology-Directed Repair.

Author information

1
Department of Genomic Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA. Electronic address: tgutschner@mdanderson.org.
2
Department of Gynecologic Oncology and Reproductive Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA; Institute of Pathology, University Hospital Heidelberg, 69120 Heidelberg, Germany.
3
Department of Genomic Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.
4
Department of Genomic Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA; Institute for Applied Cancer Science, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA; Department of Molecular and Cellular Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.
5
Department of Genomic Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA; Institute for Applied Cancer Science, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA. Electronic address: lchin@mdanderson.org.

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

PMID:
26854237
DOI:
10.1016/j.celrep.2016.01.019
[Indexed for MEDLINE]
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