1. Cell Biology
  2. Developmental Biology
Download icon

An efficient and scalable pipeline for epitope tagging in mammalian stem cells using Cas9 ribonucleoprotein

Tools and Resources
  • Cited 16
  • Views 8,055
  • Annotations
Cite this article as: eLife 2018;7:e35069 doi: 10.7554/eLife.35069

Abstract

CRISPR/Cas9 can be used for precise genetic knock-in of epitope tags into endogenous genes, simplifying experimental analysis of protein function. However, Cas9-assisted epitope tagging in primary mammalian cell cultures is often inefficient and reliant on plasmid-based selection strategies. Here we demonstrate improved knock-in efficiencies of diverse tags (V5, 3XFLAG, Myc, HA) using co-delivery of Cas9 protein pre-complexed with two-part synthetic modified RNAs (annealed crRNA:tracrRNA) and single-stranded oligodeoxynucleotide (ssODN) repair templates. Knock-in efficiencies of ~5-30%, were achieved without selection in embryonic stem (ES) cells, neural stem (NS) cells, and brain tumour-derived stem cells. Biallelic-tagged clonal lines were readily derived and used to define Olig2 chromatin-bound interacting partners. Using our novel web-based design tool, we established a 96-well format pipeline that enabled V5-tagging of 60 different transcription factors. This efficient, selection-free and scalable epitope tagging pipeline enables systematic surveys of protein expression levels, subcellular localization, and interactors across diverse mammalian stem cells.

Article and author information

Author details

  1. Pooran Singh Dewari

    MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, United Kingdom
    Competing interests
    No competing interests declared.
  2. Benjamin Southgate

    MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, United Kingdom
    Competing interests
    No competing interests declared.
  3. Katrina Mccarten

    MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, United Kingdom
    Competing interests
    No competing interests declared.
  4. German Monogarov

    German Cancer Research Center (DKFZ), Heidelberg, Germany
    Competing interests
    No competing interests declared.
  5. Eoghan O'Duibhir

    MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, United Kingdom
    Competing interests
    No competing interests declared.
  6. Niall Quinn

    Edinburgh Cancer Research UK Centre Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, United Kingdom
    Competing interests
    No competing interests declared.
  7. Ashley Tyrer

    MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, United Kingdom
    Competing interests
    No competing interests declared.
  8. Marie-Christin Leitner

    MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, United Kingdom
    Competing interests
    No competing interests declared.
  9. Colin Plumb

    MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, United Kingdom
    Competing interests
    No competing interests declared.
  10. Maria Kalantzaki

    MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, United Kingdom
    Competing interests
    No competing interests declared.
  11. Carla Blin

    MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, United Kingdom
    Competing interests
    No competing interests declared.
  12. Rebecca Finch

    MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, United Kingdom
    Competing interests
    No competing interests declared.
  13. Raul Bardini Bressan

    MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, United Kingdom
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5673-9563
  14. Gillian Morrison

    MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, United Kingdom
    Competing interests
    No competing interests declared.
  15. Ashley M Jacobi

    Integrated DNA Technologies, Inc, Coralville, United States
    Competing interests
    Ashley M Jacobi, employed by Integrated DNA Technologies (IDT), who sells reagents similar to some described herein. IDT is, however, not a publicly traded company and the authors do not own any shares or equity in IDT. No other authors have any financial interests or relationships with IDT; nor do they own any shares or equity.
  16. Mark A Behlke

    Integrated DNA Technologies, Inc, Coralville, United States
    Competing interests
    Mark A Behlke, employed by Integrated DNA Technologies (IDT), who sells reagents similar to some described herein. IDT is, however, not a publicly traded company and the authors do not own any shares or equity in IDT. No other authors have any financial interests or relationships with IDT; nor do they own any shares or equity.
  17. Alex von Kriegsheim

    Edinburgh Cancer Research UK Centre Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, United Kingdom
    Competing interests
    No competing interests declared.
  18. Simon Tomlinson

    MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, United Kingdom
    Competing interests
    No competing interests declared.
  19. Jeroen Krijgsveld

    German Cancer Research Center (DKFZ), Heidelberg, Germany
    Competing interests
    No competing interests declared.
  20. Steven M Pollard

    MRC Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, United Kingdom
    For correspondence
    steven.pollard@ed.ac.uk
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6428-0492

Funding

Cancer Research UK (A17368)

  • Pooran Singh Dewari
  • Benjamin Southgate
  • Eoghan O'Duibhir
  • Steven M Pollard

Medical Research Council (BB/M018040/1)

  • Pooran Singh Dewari
  • Steven M Pollard

Biotechnology and Biological Sciences Research Council (BB/M018040/1)

  • Pooran Singh Dewari
  • Steven M Pollard

Engineering and Physical Sciences Research Council (BB/M018040/1)

  • Pooran Singh Dewari
  • Steven M Pollard

Brain Tumour Charity (GN-000358)

  • Pooran Singh Dewari
  • Steven M Pollard

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Maarten van Lohuizen, The Netherlands Cancer Institute, Netherlands

Publication history

  1. Received: January 12, 2018
  2. Accepted: April 10, 2018
  3. Accepted Manuscript published: April 11, 2018 (version 1)
  4. Version of Record published: May 11, 2018 (version 2)

Copyright

© 2018, Dewari et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 8,055
    Page views
  • 1,534
    Downloads
  • 16
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, Scopus, PubMed Central.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)

Further reading

    1. Biochemistry and Chemical Biology
    2. Cell Biology
    Mikel Garcia-Marcos et al.
    Tools and Resources Updated

    Heterotrimeric G-proteins are signal transducers involved in mediating the action of many natural extracellular stimuli and many therapeutic agents. Non-invasive approaches to manipulate the activity of G-proteins with high precision are crucial to understand their regulation in space and time. Here, we developed LOV2GIVe, an engineered modular protein that allows the activation of heterotrimeric G-proteins with blue light. This optogenetic construct relies on a versatile design that differs from tools previously developed for similar purposes, that is metazoan opsins, which are light-activated G-protein-coupled receptors (GPCRs). Instead, LOV2GIVe consists of the fusion of a G-protein activating peptide derived from a non-GPCR regulator of G-proteins to a small plant protein domain, such that light uncages the G-protein activating module. Targeting LOV2GIVe to cell membranes allowed for light-dependent activation of Gi proteins in different experimental systems. In summary, LOV2GIVe expands the armamentarium and versatility of tools available to manipulate heterotrimeric G-protein activity.

    1. Cell Biology
    2. Plant Biology
    Madlen Stephani et al.
    Research Article Updated

    Eukaryotes have evolved various quality control mechanisms to promote proteostasis in the endoplasmic reticulum (ER). Selective removal of certain ER domains via autophagy (termed as ER-phagy) has emerged as a major quality control mechanism. However, the degree to which ER-phagy is employed by other branches of ER-quality control remains largely elusive. Here, we identify a cytosolic protein, C53, that is specifically recruited to autophagosomes during ER-stress, in both plant and mammalian cells. C53 interacts with ATG8 via a distinct binding epitope, featuring a shuffled ATG8 interacting motif (sAIM). C53 senses proteotoxic stress in the ER lumen by forming a tripartite receptor complex with the ER-associated ufmylation ligase UFL1 and its membrane adaptor DDRGK1. The C53/UFL1/DDRGK1 receptor complex is activated by stalled ribosomes and induces the degradation of internal or passenger proteins in the ER. Consistently, the C53 receptor complex and ufmylation mutants are highly susceptible to ER stress. Thus, C53 forms an ancient quality control pathway that bridges selective autophagy with ribosome-associated quality control in the ER.