title: Post-translational Regulation of Cas9 during G1 Enhances Homology-Directed Repair.
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.
authors: Department of Genomic Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA. Electronic address: tgutschner@mdanderson.org.
authors: Tony
authors: T
authors: Gutschner
authors: 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.
authors: Monika
authors: M
authors: Haemmerle
authors: Department of Genomic Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.
authors: Giannicola
authors: G
authors: Genovese
authors: 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.
authors: Giulio F
authors: GF
authors: Draetta
authors: 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.
authors: Lynda
authors: L
authors: Chin
mesh_terms: D025601:Adenomatous Polyposis Coli Protein / Q000235:genetics / Q000378:metabolism; D015703:Antigens, CD; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D000076987:CRISPR-Associated Protein 9; D064113:CRISPR-Cas Systems; D015820:Cadherins / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000235:genetics* / Q000378:metabolism; D053903:DNA Breaks, Double-Stranded; D059766:DNA End-Joining Repair; D004720:Endonucleases / Q000235:genetics* / Q000378:metabolism; D059585:G1 Phase Cell Cycle Checkpoints / Q000235:genetics*; D064248:Geminin / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D055879:Gene Knock-In Techniques / Q000379:methods; D005818:Genetic Engineering; D057809:HEK293 Cells; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D059767:Recombinational DNA Repair; D014162:Transfection; D019076:Transgenes; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism
chemical_list: C555337:APC protein, human; D025601:Adenomatous Polyposis Coli Protein; D015703:Antigens, CD; D001426:Bacterial Proteins; C513384:CDH1 protein, human; D015820:Cadherins; C577995:GMNN protein, human; D064248:Geminin; D011993:Recombinant Fusion Proteins; D004247:DNA; D044767:Ubiquitin-Protein Ligases; D000076987:CRISPR-Associated Protein 9; C000600049:Cas9 protein, Francisella novicida; D004720:Endonucleases
keywords: CRISPR; MALAT1; cell cycle; genome editing; homologous recombination; proteolysis; synthetic biology