title: Design and analysis of site-specific single-strand nicking endonucleases for gene correction.
abstract: Single-strand nicking endonucleases ("nickases") have been shown to induce homology-mediated gene correction with reduced toxicity of DNA double-strand break-producing enzymes, and nickases have been engineered from both homing endonuclease and FokI-based scaffolds. We describe the strategies used to engineer these site-specific nickases as well as the in vitro methods used to confirm their activity and specificity. Additionally, we describe the Traffic Light Reporter system, which uses a flow cytometric assay to simultaneously detect both gene repair and mutagenic nonhomologous end-joining outcomes at a single targeted site in mammalian cells. With these methods, novel nickases can be designed and tested for use in gene correction with novel target sites.
authors.affiliation: Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA, USA.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Metzger
authors.forename: Michael T
authors.initials: MT
authors.lastname: Certo
mesh_terms: D002460:Cell Line; D053904:DNA Breaks, Single-Stranded; D004260:DNA Repair; D003850:Deoxyribonuclease I / Q000378:metabolism*; D004720:Endonucleases / Q000378:metabolism; D005434:Flow Cytometry; D015870:Gene Expression; D017930:Genes, Reporter; D006801:Humans; D010957:Plasmids / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D004720:Endonucleases; D003850:Deoxyribonuclease I