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Generation of Auxin Inducible Degron (AID) Knock-in Cell Lines for Targeted Protein Degradation in Mammalian Cells

Overview
Journal STAR Protoc
Publisher Cell Press
Specialties Biology
Biomedical Engineering
Science
Date 2021 Dec 1
PMID 34849487
Citations 2
Authors
Bikash Adhikari
Ashwin Narain
Elmar Wolf
Affiliations
Soon will be listed here.
Abstract

Targeted protein degradation using degrons, such as the mini-Auxin-inducible degron (mAID), has an advantage over genetic silencing/knockout. However, the efficiency of sgRNA, homologous recombination, tedious expansion, and screening single clones makes the process of tagging endogenous proteins long and laborious. This protocol describes a practical and economical way to obtain AID-tagged endogenous proteins using CRISPR/Cas9-mediated homology-directed repair (HDR). We use the generation of endogenously AID-tagged SPT6 in U2OS cells as an example but provide sufficient details for usage in other cell types. For complete details on the use and execution of this protocol, please refer to Narain et al. (2021).

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References
1.
van Rosmalen M, Krom M, Merkx M . Tuning the Flexibility of Glycine-Serine Linkers To Allow Rational Design of Multidomain Proteins. Biochemistry. 2017; 56(50):6565-6574. PMC: 6150656. DOI: 10.1021/acs.biochem.7b00902. View
2.
Turkewitz D, Moghaddasi S, Alghalayini A, DAmario C, Ali H, Wallach M . Comparative study of His- and Non-His-tagged CLIC proteins, reveals changes in their enzymatic activity. Biochem Biophys Rep. 2021; 26:101015. PMC: 8138732. DOI: 10.1016/j.bbrep.2021.101015. View
3.
Narain A, Bhandare P, Adhikari B, Backes S, Eilers M, Dolken L . Targeted protein degradation reveals a direct role of SPT6 in RNAPII elongation and termination. Mol Cell. 2021; 81(15):3110-3127.e14. PMC: 8354102. DOI: 10.1016/j.molcel.2021.06.016. View
4.
Amen T, Kaganovich D . Quantitative photoconversion analysis of internal molecular dynamics in stress granules and other membraneless organelles in live cells. STAR Protoc. 2020; 1(3):100217. PMC: 7757677. DOI: 10.1016/j.xpro.2020.100217. View
5.
Florea B, Meaney C, Junginger H, Borchard G . Transfection efficiency and toxicity of polyethylenimine in differentiated Calu-3 and nondifferentiated COS-1 cell cultures. AAPS PharmSci. 2002; 4(3):E12. PMC: 2751351. DOI: 10.1208/ps040312. View