title: Use of the HPRT gene to study nuclease-induced DNA double-strand break repair.
abstract: Understanding the mechanisms of chromosomal double-strand break repair (DSBR) provides insight into genome instability, oncogenesis and genome engineering, including disease gene correction. Research into DSBR exploits rare-cutting endonucleases to cleave exogenous reporter constructs integrated into the genome. Multiple reporter constructs have been developed to detect various DSBR pathways. Here, using a single endogenous reporter gene, the X-chromosomal disease gene encoding hypoxanthine phosphoribosyltransferase (HPRT), we monitor the relative utilization of three DSBR pathways following cleavage by I-SceI or CRISPR/Cas9 nucleases. For I-SceI, our estimated frequencies of accurate or mutagenic non-homologous end-joining and gene correction by homologous recombination are 4.1, 1.5 and 0.16%, respectively. Unexpectedly, I-SceI and Cas9 induced markedly different DSBR profiles. Also, using an I-SceI-sensitive HPRT minigene, we show that gene correction is more efficient when using long double-stranded DNA than single- or double-stranded oligonucleotides. Finally, using both endogenous HPRT and exogenous reporters, we validate novel cell cycle phase-specific I-SceI derivatives for investigating cell cycle variations in DSBR. The results obtained using these novel approaches provide new insights into template design for gene correction and the relationships between multiple DSBR pathways at a single endogenous disease gene.
authors.affiliation: Gene Targeting Group, Centre for Haematology, Imperial College Faculty of Medicine, London W120NN, UK and.
authors.forename: Polly
authors.initials: P
authors.lastname: Gravells
authors.affiliation: CRUK MRC Oxford Institute for Radiation Oncology, Department of Oncology, University of Oxford, Oxford OX3 7DQ, UK.
authors.forename: Sara
authors.initials: S
authors.lastname: Ahrabi
authors.affiliation: Gene Targeting Group, Centre for Haematology, Imperial College Faculty of Medicine, London W120NN, UK and.
authors.forename: Rajani K
authors.initials: RK
authors.lastname: Vangala
authors.affiliation: Gene Targeting Group, Centre for Haematology, Imperial College Faculty of Medicine, London W120NN, UK and.
authors.forename: Kazunori
authors.initials: K
authors.lastname: Tomita
authors.affiliation: Gene Targeting Group, Centre for Haematology, Imperial College Faculty of Medicine, London W120NN, UK and.
authors.forename: James T
authors.initials: JT
authors.lastname: Brash
authors.affiliation: Gene Targeting Group, Centre for Haematology, Imperial College Faculty of Medicine, London W120NN, UK and.
authors.forename: Lena A
authors.initials: LA
authors.lastname: Brustle
authors.affiliation: Gene Targeting Group, Centre for Haematology, Imperial College Faculty of Medicine, London W120NN, UK and.
authors.forename: Christopher
authors.initials: C
authors.lastname: Chung
authors.affiliation: Gene Targeting Group, Centre for Haematology, Imperial College Faculty of Medicine, London W120NN, UK and.
authors.forename: Julia M
authors.initials: JM
authors.lastname: Hong
authors.affiliation: Gene Targeting Group, Centre for Haematology, Imperial College Faculty of Medicine, London W120NN, UK and.
authors.forename: Aikaterini
authors.initials: A
authors.lastname: Kaloudi
authors.affiliation: CRUK MRC Oxford Institute for Radiation Oncology, Department of Oncology, University of Oxford, Oxford OX3 7DQ, UK.
authors.forename: Timothy C
authors.initials: TC
authors.lastname: Humphrey
authors.affiliation: Gene Targeting Group, Centre for Haematology, Imperial College Faculty of Medicine, London W120NN, UK and andy.porter@imperial.ac.uk.
authors.forename: Andrew C G
authors.initials: AC
authors.lastname: Porter
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism; D000076987:CRISPR-Associated Protein 9; D064113:CRISPR-Cas Systems; D002453:Cell Cycle; D045744:Cell Line, Tumor; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D004720:Endonucleases / Q000378:metabolism*; D017930:Genes, Reporter; D006367:HeLa Cells; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics*; D051379:Mice; D016296:Mutagenesis; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D029701:Saccharomyces cerevisiae Proteins; D007041:Hypoxanthine Phosphoribosyltransferase; D000076987:CRISPR-Associated Protein 9; C000606107:Cas9 endonuclease Streptococcus pyogenes; D004720:Endonucleases; C037725:SCEI protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific