title: CRISPR-Cas9 mediated genetic engineering for the purification of the endogenous integrator complex from mammalian cells.
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.
authors.affiliation: Department of Biochemistry and Molecular Biology, The University of Texas Medical Branch, Galveston, TX, USA. Electronic address: dabailla@utmb.edu.
authors.forename: David
authors.initials: D
authors.lastname: Baillat
authors.affiliation: Department of Biochemistry and Molecular Biology, The University of Texas Medical Branch, Galveston, TX, USA.
authors.forename: William K
authors.initials: WK
authors.lastname: Russell
authors.affiliation: Department of Biochemistry and Molecular Biology, The University of Texas Medical Branch, Galveston, TX, USA. Electronic address: ejwagner@utmb.edu.
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Wagner
mesh_terms: D064113:CRISPR-Cas Systems; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D004722:Endoribonucleases; D000072669:Gene Editing / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D009687:Nuclear Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D002352:Carrier Proteins; C581361:INTS4 protein, human; D009687:Nuclear Proteins; D004722:Endoribonucleases; C505818:INTS11 protein, human
keywords: CRISPR-mediated purification; Integrator complex; Mass spectrometry