Microhomology-based CRISPR tagging tools for protein tracking, purification, and depletion.

Autor: Lin DW; Departments of Biological Chemistry and., Chung BP; Departments of Biological Chemistry and., Huang JW; Departments of Biological Chemistry and., Wang X; Physiology and Biophysics, University of California, Irvine, California 92617., Huang L; Physiology and Biophysics, University of California, Irvine, California 92617., Kaiser P; Departments of Biological Chemistry and. Electronic address: pkaiser@uci.edu.
Jazyk: angličtina
Zdroj: The Journal of biological chemistry [J Biol Chem] 2019 Jul 12; Vol. 294 (28), pp. 10877-10885. Date of Electronic Publication: 2019 May 28.
DOI: 10.1074/jbc.RA119.008422
Abstrakt: Work in yeast models has benefitted tremendously from the insertion of epitope or fluorescence tags at the native gene locus to study protein function and behavior under physiological conditions. In contrast, work in mammalian cells largely relies on overexpression of tagged proteins because high-quality antibodies are only available for a fraction of the mammalian proteome. CRISPR/Cas9-mediated genome editing has recently emerged as a powerful genome-modifying tool that can also be exploited to insert various tags and fluorophores at gene loci to study the physiological behavior of proteins in most organisms, including mammals. Here we describe a versatile toolset for rapid tagging of endogenous proteins. The strategy utilizes CRISPR/Cas9 and microhomology-mediated end joining repair for efficient tagging. We provide tools to insert 3×HA, His 6 FLAG, His 6 -Biotin-TEV-RGSHis 6 , mCherry, GFP, and the auxin-inducible degron tag for compound-induced protein depletion. This approach and the developed tools should greatly facilitate functional analysis of proteins in their native environment.
(© 2019 Lin et al.)
Databáze: MEDLINE
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