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

Elena Shvets, Carolina Mendoza-Topaz

Methods in molecular biology (Clifton, N.J.) 2020

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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.

Citation

Elena Shvets, Carolina Mendoza-Topaz. Tagging and Deleting of Endogenous Caveolar Components Using CRISPR/Cas9 Technology. Methods in molecular biology (Clifton, N.J.). 2020;2169:149-166