Zinc-finger nucleases (ZFNs) drive efficient genome editing by introducing a double-strand break into the targeted gene. Cleavage is induced when two custom-designed ZFNs heterodimerize upon binding DNA to form a catalytically active nuclease complex. The importance of this dimerization event for subsequent cleavage activity has stimulated efforts to engineer the nuclease interface to prevent undesired homodimerization. Here we report the development and application of a yeast-based selection system designed to functionally interrogate the ZFN dimer interface. We identified critical residues involved in dimerization through the isolation of cold-sensitive nuclease domains. We used these residues to engineer ZFNs that have superior cleavage activity while suppressing homodimerization. The improvements were portable to orthogonal domains, allowing the concomitant and independent cleavage of two loci using two different ZFN pairs. These ZFN architectures provide a general means for obtaining highly efficient and specific genome modification.
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http://dx.doi.org/10.1038/nmeth.1539 | DOI Listing |
Nucleic Acids Res
January 2025
Research Center for Life Sciences Computing, Zhejiang Lab, Kechuang Avenue, Yuhang District, Hangzhou, Zhejiang, 311121, China.
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Fraunhofer Institute for Translational Medicine and Pharmacology ITMP, Discovery Research ScreeningPort, Schnackenburgallee 114, 22525 Hamburg, Germany.
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View Article and Find Full Text PDFNarra J
December 2024
Animal Research Facilities, Indonesia Medical Education and Research Institute (IMERI), Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia.
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View Article and Find Full Text PDFNat Chem Biol
January 2025
Department of Chemical Engineering, Stanford University, Stanford, CA, USA.
Synthetic circuits that regulate protein secretion in human cells could support cell-based therapies by enabling control over local environments. Although protein-level circuits enable such potential clinical applications, featuring orthogonality and compactness, their non-human origin poses a potential immunogenic risk. In this study, we developed Humanized Drug Induced Regulation of Engineered CyTokines (hDIRECT) as a platform to control cytokine activity exclusively using human-derived proteins.
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