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

Helene Damhofer; Aliaksandra Radzisheuskaya; Kristian Helin

doi:10.1016/j.xpro.2021.100575

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

STAR Protoc. 2021 Jun 2;2(2):100575. doi: 10.1016/j.xpro.2021.100575. eCollection 2021 Jun 18.

Authors

Helene Damhofer^{1

2

3}, Aliaksandra Radzisheuskaya^{1

2

3}, Kristian Helin^{1

2

3}

Affiliations

¹ Cell Biology Program and Center for Epigenetics Research, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA.
² Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Copenhagen N 2200, Denmark.
³ The Novo Nordisk Foundation Center for Stem Cell Biology (Danstem), University of Copenhagen, University of Copenhagen, Copenhagen N 2200, Denmark.

Abstract

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).

Keywords: CRISPR; Genetics; Molecular Biology.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

Animals
Cell Line
Chromosome Mapping*
Gene Knock-In Techniques*
Genetic Vectors
Humans
Mice
Mouse Embryonic Stem Cells / metabolism
Plasmids

Grants and funding

P30 CA008748/CA/NCI NIH HHS/United States