CRISPR-Cas9 mediated genetic engineering for the purification of the endogenous integrator complex from mammalian cells

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

The Integrator Complex (INT) is a large multi-subunit protein complex, containing at least 14 subunits and a host of associated factors. These protein components have been established through pulldowns of overexpressed epitope tagged subunits or by using antibodies raised against specific subunits. Here, we utilize CRISPR/Cas9 gene editing technology to introduce N-terminal FLAG epitope tags into the endogenous genes that encode Integrator subunit 4 and 11 within HEK293T cells. We provide specific details regarding design, approaches for facile screening, and our observed frequency of successful recombination. Finally, using silver staining, Western blotting and LC-MS/MS we compare the components of INT of purifications from CRISPR derived lines to 293T cells overexpressing FLAG-INTS11 to define a highly resolved constituency of mammalian INT.

Original languageEnglish (US)
Pages (from-to)101-108
Number of pages8
JournalProtein Expression and Purification
Volume128
DOIs
StatePublished - Dec 1 2016

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Clustered Regularly Interspaced Short Palindromic Repeats
Genetic Engineering
Epitopes
Silver Staining
HEK293 Cells
Protein Subunits
Genetic Recombination
Western Blotting
Technology
Antibodies
Genes
Proteins

Keywords

  • CRISPR-mediated purification
  • Integrator complex
  • Mass spectrometry

ASJC Scopus subject areas

  • Biotechnology

Cite this

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abstract = "The Integrator Complex (INT) is a large multi-subunit protein complex, containing at least 14 subunits and a host of associated factors. These protein components have been established through pulldowns of overexpressed epitope tagged subunits or by using antibodies raised against specific subunits. Here, we utilize CRISPR/Cas9 gene editing technology to introduce N-terminal FLAG epitope tags into the endogenous genes that encode Integrator subunit 4 and 11 within HEK293T cells. We provide specific details regarding design, approaches for facile screening, and our observed frequency of successful recombination. Finally, using silver staining, Western blotting and LC-MS/MS we compare the components of INT of purifications from CRISPR derived lines to 293T cells overexpressing FLAG-INTS11 to define a highly resolved constituency of mammalian INT.",
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