Abstract
Zinc-finger nucleases (ZFNs) are programmable nucleases that have opened the door to the genome editing era. The construction of ZFNs recognizing a target sequence of interest is laborious, and has not been widely used recently. However, key ZFN patents are expiring over the next 2–4 years, enabling a wide range of deployments for clinical and industrial applications. This article introduces a ZFN construction protocol that uses bacterial one-hybrid (B1H) selection and single-stranded annealing (SSA) assay.
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Acknowledgment
We thank Dr. Scot Wolfe for providing the pH3U3-mcs reporter vector and US0ΔhisBΔpyrFΔrpoZ bacterial strain (Addgene plasmids 12609 and 18049, respectively). This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (15 K18467,16H01407) and by the JST PRESTO program to H.O.
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Ochiai, H., Yamamoto, T. (2017). Construction and Evaluation of Zinc Finger Nucleases. In: Hatada, I. (eds) Genome Editing in Animals. Methods in Molecular Biology, vol 1630. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7128-2_1
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DOI: https://doi.org/10.1007/978-1-4939-7128-2_1
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Publisher Name: Humana Press, New York, NY
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Online ISBN: 978-1-4939-7128-2
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