Generation of locus-specific degradable tag knock-ins in mouse and human cell lines

Generation of locus-specific degradable tag knock-ins in mouse and human cell lines

Summary: Protein degradation technologies represent a powerful functional genomics tool, allowing fast and controllable target protein depletion. Establishing these systems requires a knock-in of the degradation tag into both endogenous target gene alleles. Here, we provide a step-by-step protocol f...

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Bibliographic Details
Main Authors: Helene Damhofer, Aliaksandra Radzisheuskaya, Kristian Helin
Format: Article
Language:English
Published: Elsevier 2021-06-01
Series:STAR Protocols
Subjects:
CRISPR
Genetics
Molecular Biology
Online Access:http://www.sciencedirect.com/science/article/pii/S2666166721002823
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spelling doaj-a1859a67b9bc414588ca5376f7d5d55f2021-06-21T04:25:46ZengElsevierSTAR Protocols2666-16672021-06-0122100575Generation of locus-specific degradable tag knock-ins in mouse and human cell linesHelene Damhofer0Aliaksandra Radzisheuskaya1Kristian Helin2Cell Biology Program and Center for Epigenetics Research, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA; Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Copenhagen N 2200, Denmark; The Novo Nordisk Foundation Center for Stem Cell Biology (Danstem), University of Copenhagen, University of Copenhagen, Copenhagen N 2200, Denmark; Corresponding authorCell Biology Program and Center for Epigenetics Research, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA; Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Copenhagen N 2200, Denmark; The Novo Nordisk Foundation Center for Stem Cell Biology (Danstem), University of Copenhagen, University of Copenhagen, Copenhagen N 2200, DenmarkCell Biology Program and Center for Epigenetics Research, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA; Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Copenhagen N 2200, Denmark; The Novo Nordisk Foundation Center for Stem Cell Biology (Danstem), University of Copenhagen, University of Copenhagen, Copenhagen N 2200, Denmark; Corresponding authorSummary: Protein degradation technologies represent a powerful functional genomics tool, allowing fast and controllable target protein depletion. Establishing these systems requires a knock-in of the degradation tag into both endogenous target gene alleles. Here, we provide a step-by-step protocol for the efficient generation of biallelic degradation tag knock-ins in mouse and human cell lines using CRISPR-Cas9. We use knockin of an endogenous Kansl3 degradation tag in mouse embryonic stem (ES) cells as an example but provide modifications for application in other cell types.For complete details on the use and execution of this protocol, please refer to Radzisheuskaya et al. (2021).http://www.sciencedirect.com/science/article/pii/S2666166721002823CRISPRGeneticsMolecular Biology
collection DOAJ
language English
format Article
sources DOAJ
author Helene Damhofer
Aliaksandra Radzisheuskaya
Kristian Helin
spellingShingle Helene Damhofer
Aliaksandra Radzisheuskaya
Kristian Helin
Generation of locus-specific degradable tag knock-ins in mouse and human cell lines
STAR Protocols
CRISPR
Genetics
Molecular Biology
author_facet Helene Damhofer
Aliaksandra Radzisheuskaya
Kristian Helin
author_sort Helene Damhofer
title Generation of locus-specific degradable tag knock-ins in mouse and human cell lines
title_short Generation of locus-specific degradable tag knock-ins in mouse and human cell lines
title_full Generation of locus-specific degradable tag knock-ins in mouse and human cell lines
title_fullStr Generation of locus-specific degradable tag knock-ins in mouse and human cell lines
title_full_unstemmed Generation of locus-specific degradable tag knock-ins in mouse and human cell lines
title_sort generation of locus-specific degradable tag knock-ins in mouse and human cell lines
publisher Elsevier
series STAR Protocols
issn 2666-1667
publishDate 2021-06-01
description Summary: Protein degradation technologies represent a powerful functional genomics tool, allowing fast and controllable target protein depletion. Establishing these systems requires a knock-in of the degradation tag into both endogenous target gene alleles. Here, we provide a step-by-step protocol for the efficient generation of biallelic degradation tag knock-ins in mouse and human cell lines using CRISPR-Cas9. We use knockin of an endogenous Kansl3 degradation tag in mouse embryonic stem (ES) cells as an example but provide modifications for application in other cell types.For complete details on the use and execution of this protocol, please refer to Radzisheuskaya et al. (2021).
topic CRISPR
Genetics
Molecular Biology
url http://www.sciencedirect.com/science/article/pii/S2666166721002823
work_keys_str_mv AT helenedamhofer generationoflocusspecificdegradabletagknockinsinmouseandhumancelllines
AT aliaksandraradzisheuskaya generationoflocusspecificdegradabletagknockinsinmouseandhumancelllines
AT kristianhelin generationoflocusspecificdegradabletagknockinsinmouseandhumancelllines
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