Protocol for CRISPR-based endogenous protein tagging in mammalian cells.
| Autor: | Xiao YX; Program in Genetics and Genome Biology, The Hospital for Sick Children, Toronto, ON, Canada; Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada., Wei J; Program in Genetics and Genome Biology, The Hospital for Sick Children, Toronto, ON, Canada; Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada., Moffat J; Program in Genetics and Genome Biology, The Hospital for Sick Children, Toronto, ON, Canada; Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada; Institute for Biomedical Engineering, University of Toronto, Toronto, ON, Canada. Electronic address: jason.moffat@sickkids.ca. |
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| Jazyk: | angličtina |
| Zdroj: | STAR protocols [STAR Protoc] 2024 Dec 20; Vol. 5 (4), pp. 103404. Date of Electronic Publication: 2024 Oct 19. |
| DOI: | 10.1016/j.xpro.2024.103404 |
| Abstrakt: | Tracking the localization and proximal interaction partners of endogenous proteins provides valuable functional insight. Here, we present a protocol for CRISPR-based endogenous protein tagging in mammalian cells. We describe steps for endogenously tagging human TSC22D2 and MAP4, including designing Cas9 and Cas12a guides for knockin, modularized repair template design and cloning, and procedures for lipid transfection and electroporation. This protocol accommodates Cas nucleases in plasmid expression or ribonucleoprotein complex (RNP) formats. This "endo-tagging" approach offers flexibility and broad applicability. For complete details on the use and execution of this protocol, please refer to Xiao et al. 1 . Competing Interests: Declaration of interests The authors declare no competing interests. (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.) |
| Databáze: | MEDLINE |
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