title: Nuclear domain 'knock-in' screen for the evaluation and identification of small molecule enhancers of CRISPR-based genome editing.
abstract: CRISPR is a genome-editing platform that makes use of the bacterially-derived endonuclease Cas9 to introduce DNA double-strand breaks at precise locations in the genome using complementary guide RNAs. We developed a nuclear domain knock-in screen, whereby the insertion of a gene encoding the green fluorescent protein variant Clover is inserted by Cas9-mediated homology directed repair (HDR) within the first exon of genes that are required for the structural integrity of subnuclear domains such as the nuclear lamina and promyelocytic leukemia nuclear bodies (PML NBs). Using this approach, we compared strategies for enhancing CRISPR-mediated HDR, focusing on known genes and small molecules that impact non-homologous end joining (NHEJ) and homologous recombination (HR). Ultimately, we identified the small molecule RS-1 as a potent enhancer of CRISPR-based genome editing, enhancing HDR 3- to 6-fold depending on the locus and transfection method. We also characterized U2OS human osteosarcoma cells expressing Clover-tagged PML and demonstrate that this strategy generates cell lines with PML NBs that are structurally and functionally similar to bodies in the parental cell line. Thus, the nuclear domain knock-in screen that we describe provides a simple means of rapidly evaluating methods and small molecules that have the potential to enhance Cas9-mediated HDR.
authors.affiliation: Department of Pathology, Dalhousie University, Halifax, Nova Scotia, B3H 4R2, Canada.
authors.forename: Jordan
authors.initials: J
authors.lastname: Pinder
authors.affiliation: Department of Pathology, Dalhousie University, Halifax, Nova Scotia, B3H 4R2, Canada.
authors.forename: Jayme
authors.initials: J
authors.lastname: Salsman
authors.affiliation: Department of Pathology, Dalhousie University, Halifax, Nova Scotia, B3H 4R2, Canada Department of Biochemistry & Molecular Biology, Dalhousie University, Halifax, Nova Scotia, B3H 4R2, Canada Beatrice Hunter Cancer Research Institute, Halifax, Nova Scotia, B3H 4R2, Canada dellaire@dal.ca.
authors.forename: Graham
authors.initials: G
authors.lastname: Dellaire
mesh_terms: D019313:BRCA1 Protein / Q000378:metabolism; D001549:Benzamides / Q000494:pharmacology; D064113:CRISPR-Cas Systems; D045744:Cell Line, Tumor; D053903:DNA Breaks, Double-Stranded; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D005434:Flow Cytometry; D055879:Gene Knock-In Techniques / Q000379:methods*; D016678:Genome; D049452:Green Fluorescent Proteins / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D009687:Nuclear Proteins / Q000032:analysis / Q000235:genetics; D009841:Oligonucleotides; D011743:Pyrimidines / Q000494:pharmacology; D012313:RNA / Q000378:metabolism; D051135:Rad51 Recombinase / Q000378:metabolism; D059767:Recombinational DNA Repair; D012545:Schiff Bases / Q000494:pharmacology; D013449:Sulfonamides / Q000494:pharmacology
chemical_list: C000605335:3-((benzylamino)sulfonyl)-4-bromo-N-(4-bromophenyl)benzamide; C578537:5,6-bis(benzylideneamino)-2-mercaptopyrimidin-4-ol; D019313:BRCA1 Protein; D001549:Benzamides; D009687:Nuclear Proteins; D009841:Oligonucleotides; D011743:Pyrimidines; D012545:Schiff Bases; D013449:Sulfonamides; D049452:Green Fluorescent Proteins; D012313:RNA; D051135:Rad51 Recombinase; D004706:Endodeoxyribonucleases