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.
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Chen, F., Pruett-Miller, S.M., Davis, G.D. (2015). Gene Editing Using ssODNs with Engineered Endonucleases. In: Pruett-Miller, S. (eds) Chromosomal Mutagenesis. Methods in Molecular Biology, vol 1239. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1862-1_14
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DOI: https://doi.org/10.1007/978-1-4939-1862-1_14
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