Abstract
Zinc finger nucleases (ZFNs) are programmable nucleases that have contributed significantly to past genome-editing research. They are now utilized much less owing to the advent of transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats and CRISPR-associated protein system (CRISPR-Cas). These new methods allow for easier generation of reagents that target genomic sequences of interest and are less labor-intensive than ZFNs at targeting desired sequences. However, fundamental ZFN patents have expired, enabling a wide range of their distribution for clinical and industrial applications. This article introduces a ZFN construction protocol that uses bacterial one-hybrid (B1H) selection and single-strand annealing (SSA) assay.
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References
Urnov FD, Rebar EJ, Holmes MC et al (2010) Genome editing with engineered zinc finger nucleases. Nat Rev Genet 11:636–646. https://doi.org/10.1038/nrg2842
Carroll D (2014) Genome engineering with targetable nucleases. Annu Rev Biochem 83:409–439. https://doi.org/10.1146/annurev-biochem-060713-035418
Scott CT (2005) The zinc finger nuclease monopoly. Nat Biotechnol 23:915–918. https://doi.org/10.1038/nbt0805-915
Ochiai H, Fujita K, Suzuki K-I et al (2010) Targeted mutagenesis in the sea urchin embryo using zinc-finger nucleases. Genes Cells 15:875–885. https://doi.org/10.1111/j.1365-2443.2010.01425.x
Wolfe SA, Nekludova L, Pabo CO (2000) DNA recognition by Cys2His2 zinc finger proteins. Annu Rev Biophys Biomol Struct 29:183–212. https://doi.org/10.1146/annurev.biophys.29.1.183
Klug A (2010) The discovery of zinc fingers and their applications in gene regulation and genome manipulation. Annu Rev Biochem 79:213–231. https://doi.org/10.1146/annurev-biochem-010909-095056
Sakuma T, Hosoi S, Woltjen K et al (2013) Efficient TALEN construction and evaluation methods for human cell and animal applications. Genes Cells 18:315–326. https://doi.org/10.1111/gtc.12037
Wright DA, Thibodeau-Beganny S, Sander JD et al (2006) Standardized reagents and protocols for engineering zinc finger nucleases by modular assembly. Nat Protoc 1:1637–1652. https://doi.org/10.1038/nprot.2006.259
Meng X, Wolfe SA (2006) Identifying DNA sequences recognized by a transcription factor using a bacterial one-hybrid system. Nat Protoc 1:30–45. https://doi.org/10.1038/nprot.2006.6
Carroll D, Morton JJ, Beumer KJ, Segal DJ (2006) Design, construction and in vitro testing of zinc finger nucleases. Nat Protoc 1:1329–1341. https://doi.org/10.1038/nprot.2006.231
Ramirez CL, Foley JE, Wright DA et al (2008) Unexpected failure rates for modular assembly of engineered zinc fingers. Nat Methods 5:374–375. https://doi.org/10.1038/nmeth0508-374
Alwin S, Gere MB, Guhl E et al (2005) Custom zinc-finger nucleases for use in human cells. Mol Ther 12:610–617. https://doi.org/10.1016/j.ymthe.2005.06.094
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 (15K18467, 16H01407) and by the JST PRESTO program to H.O.
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Ochiai, H., Yamamoto, T. (2023). Construction and Evaluation of Zinc Finger Nucleases. In: Hatada, I. (eds) Genome Editing in Animals. Methods in Molecular Biology, vol 2637. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3016-7_1
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DOI: https://doi.org/10.1007/978-1-0716-3016-7_1
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