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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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 |
_version_ |
1721368902022201344 |