title: Gene editing using ssODNs with engineered endonucleases.
abstract: Gene editing using engineered endonucleases, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 nucleases, requires the creation of a targeted, chromosomal DNA double-stranded break (DSB). In mammalian cells, these DSBs are typically repaired by one of the two major DNA repair pathways: nonhomologous end joining (NHEJ) or homology-directed repair (HDR). NHEJ is an error-prone repair process that can result in a wide range of end-joining events that leads to somewhat random mutations at the site of DSB. HDR is a precise repair pathway that can utilize either an endogenous or exogenous piece of homologous DNA as a template or "donor" for repair. Traditional gene editing via HDR has relied on the co-delivery of a targeted, engineered endonuclease and a circular plasmid donor construct. More recently, it has been shown that single-stranded oligodeoxynucleotides (ssODNs) can also serve as DNA donors and thus obviate the more laborious and time-consuming plasmid vector construction process. Here we describe the use of ssODNs for making defined genome modifications in combination with engineered endonucleases.
authors.affiliation: Sigma-Aldrich Biotechnology, 2909 Laclede Ave., St. Louis, MO, 63103, USA.
authors.forename: Fuqiang
authors.initials: F
authors.lastname: Chen
authors.forename: Shondra M
authors.initials: SM
authors.lastname: Pruett-Miller
authors.forename: Gregory D
authors.initials: GD
authors.lastname: Davis
mesh_terms: D003001:Cloning, Molecular / Q000379:methods; D053903:DNA Breaks, Double-Stranded; D059766:DNA End-Joining Repair; D004277:DNA, Single-Stranded; D004720:Endonucleases / Q000235:genetics*; D005434:Flow Cytometry / Q000379:methods; D015870:Gene Expression; D018390:Gene Targeting / Q000379:methods; D005818:Genetic Engineering / Q000379:methods*; D040741:Genetic Load; D005822:Genetic Vectors / Q000235:genetics; D016254:Mutagenesis, Insertional; D009838:Oligodeoxyribonucleotides; D038744:Ribosomal Protein S6 Kinases, 90-kDa / Q000235:genetics; D059010:Single-Cell Analysis / Q000379:methods; D014162:Transfection
chemical_list: D004277:DNA, Single-Stranded; D009838:Oligodeoxyribonucleotides; D038744:Ribosomal Protein S6 Kinases, 90-kDa; C069330:ribosomal protein S6 kinase, 90kDa, polypeptide 3; D004720:Endonucleases