Generation of auxin inducible degron (AID) knock-in cell lines for targeted protein degradation in mammalian cells

Summary: 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 pr...

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Autores principales:	Bikash Adhikari, Ashwin Narain, Elmar Wolf
Formato:	article
Lenguaje:	EN
Publicado:	Elsevier 2021
Materias:	Cell Biology Molecular Biology CRISPR Science (General) Q1-390
Acceso en línea:	https://doaj.org/article/eabd39a47e3e4e508f16a6a2b0613cc0
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spelling	oai:doaj.org-article:eabd39a47e3e4e508f16a6a2b0613cc02021-11-22T04:30:56ZGeneration of auxin inducible degron (AID) knock-in cell lines for targeted protein degradation in mammalian cells2666-166710.1016/j.xpro.2021.100949https://doaj.org/article/eabd39a47e3e4e508f16a6a2b0613cc02021-12-01T00:00:00Zhttp://www.sciencedirect.com/science/article/pii/S2666166721006559https://doaj.org/toc/2666-1667Summary: 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).Bikash AdhikariAshwin NarainElmar WolfElsevierarticleCell BiologyMolecular BiologyCRISPRScience (General)Q1-390ENSTAR Protocols, Vol 2, Iss 4, Pp 100949- (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Cell Biology Molecular Biology CRISPR Science (General) Q1-390
spellingShingle	Cell Biology Molecular Biology CRISPR Science (General) Q1-390 Bikash Adhikari Ashwin Narain Elmar Wolf Generation of auxin inducible degron (AID) knock-in cell lines for targeted protein degradation in mammalian cells
description	Summary: 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).
format	article
author	Bikash Adhikari Ashwin Narain Elmar Wolf
author_facet	Bikash Adhikari Ashwin Narain Elmar Wolf
author_sort	Bikash Adhikari
title	Generation of auxin inducible degron (AID) knock-in cell lines for targeted protein degradation in mammalian cells
title_short	Generation of auxin inducible degron (AID) knock-in cell lines for targeted protein degradation in mammalian cells
title_full	Generation of auxin inducible degron (AID) knock-in cell lines for targeted protein degradation in mammalian cells
title_fullStr	Generation of auxin inducible degron (AID) knock-in cell lines for targeted protein degradation in mammalian cells
title_full_unstemmed	Generation of auxin inducible degron (AID) knock-in cell lines for targeted protein degradation in mammalian cells
title_sort	generation of auxin inducible degron (aid) knock-in cell lines for targeted protein degradation in mammalian cells
publisher	Elsevier
publishDate	2021
url	https://doaj.org/article/eabd39a47e3e4e508f16a6a2b0613cc0
work_keys_str_mv	AT bikashadhikari generationofauxininducibledegronaidknockincelllinesfortargetedproteindegradationinmammaliancells AT ashwinnarain generationofauxininducibledegronaidknockincelllinesfortargetedproteindegradationinmammaliancells AT elmarwolf generationofauxininducibledegronaidknockincelllinesfortargetedproteindegradationinmammaliancells
_version_	1718418196169490432

Generation of auxin inducible degron (AID) knock-in cell lines for targeted protein degradation in mammalian cells

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