Tagging and Deleting of Endogenous Caveolar Components Using CRISPR/Cas9 Technology

Methods Mol Biol. 2020:2169:149-166. doi: 10.1007/978-1-0716-0732-9_14.

Abstract

Here, we describe how to utilize CRISPR/Cas9 technology in the generation of tissue culture cells with fluorescently tagged caveolar components as well as cells deleted of endogenous caveolar components. As one example, we will describe tagging of EHD2, caveolar neck protein, with Green Fluorescent protein (eGFP) from endogenous loci (knock-in, KI). As another example, we will describe deletion (knock-out, KO) of Caveolin1 (Cav1), an essential caveolar component in NIH/3T3 cells. In both instances, the modifications were achieved by using Cas9 delivery on plasmid DNA by electroporation and by utilizing FACS cell sorting for selection or enrichment of edited population of cells. We also provide a list with tested gRNA sequences to successfully produce KI and KO of other caveolar components.

Keywords: Caveolae; Caveolin1; Cavin1; EHD2; Genome-editing; Knock-in; Knock-out; NIH/3T3 cells.

MeSH terms

  • Animals
  • CRISPR-Associated Protein 9 / genetics
  • CRISPR-Associated Protein 9 / metabolism
  • CRISPR-Cas Systems*
  • Carrier Proteins / genetics*
  • Carrier Proteins / metabolism
  • Caveolae / metabolism
  • Caveolin 1 / genetics*
  • Caveolin 1 / metabolism
  • Cloning, Molecular / methods
  • Electroporation / methods
  • Flow Cytometry
  • Fluorescent Antibody Technique / methods
  • Gene Editing / methods*
  • Gene Knock-In Techniques / methods*
  • Gene Knockout Techniques / methods*
  • Green Fluorescent Proteins / genetics
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism*
  • Mice
  • NIH 3T3 Cells
  • Plasmids / genetics
  • RNA-Binding Proteins / genetics
  • RNA-Binding Proteins / metabolism*

Substances

  • Carrier Proteins
  • Caveolin 1
  • Cavin1 protein, mouse
  • EHD2 protein, mouse
  • Membrane Proteins
  • RNA-Binding Proteins
  • Green Fluorescent Proteins
  • CRISPR-Associated Protein 9