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.
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Acknowledgments
This work was supported by fellowships from National Institute of Health to MJM (CA009657 and CA009503) and MTC (GM007270). We thank Ryo Takeuchi for the purified I-AniIY2.
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Metzger, M.J., Certo, M.T. (2014). Design and Analysis of Site-Specific Single-Strand Nicking Endonucleases for Gene Correction. In: Storici, F. (eds) Gene Correction. Methods in Molecular Biology, vol 1114. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-761-7_15
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DOI: https://doi.org/10.1007/978-1-62703-761-7_15
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Publisher Name: Humana Press, Totowa, NJ
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Online ISBN: 978-1-62703-761-7
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