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

David Baillat; William K. Russell; Eric J. Wagner

doi:10.1016/j.pep.2016.08.011

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

David Baillat, William K. Russell, Eric J. Wagner

Biochemistry & Molecular Biology

Research output: Contribution to journal › Article › peer-review

8 Scopus citations

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 language	English (US)
Pages (from-to)	101-108
Number of pages	8
Journal	Protein Expression and Purification
Volume	128
DOIs	https://doi.org/10.1016/j.pep.2016.08.011
State	Published - Dec 1 2016

Keywords

CRISPR-mediated purification
Integrator complex
Mass spectrometry

ASJC Scopus subject areas

Biotechnology

Access to Document

10.1016/j.pep.2016.08.011

Cite this

@article{8b8dfa1b58bf4be0baf6942ca7887b7c,

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.",

keywords = "CRISPR-mediated purification, Integrator complex, Mass spectrometry",

author = "David Baillat and Russell, {William K.} and Wagner, {Eric J.}",

note = "Publisher Copyright: {\textcopyright} 2016 Elsevier Inc.",

year = "2016",

month = dec,

day = "1",

doi = "10.1016/j.pep.2016.08.011",

language = "English (US)",

volume = "128",

pages = "101--108",

journal = "Protein Expression and Purification",

issn = "1046-5928",

publisher = "Academic Press Inc.",

}

TY - JOUR

T1 - CRISPR-Cas9 mediated genetic engineering for the purification of the endogenous integrator complex from mammalian cells

AU - Baillat, David

AU - Russell, William K.

AU - Wagner, Eric J.

PY - 2016/12/1

Y1 - 2016/12/1

N2 - 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.

AB - 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.

KW - CRISPR-mediated purification

KW - Integrator complex

KW - Mass spectrometry

UR - http://www.scopus.com/inward/record.url?scp=84984856953&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84984856953&partnerID=8YFLogxK

U2 - 10.1016/j.pep.2016.08.011

DO - 10.1016/j.pep.2016.08.011

M3 - Article

C2 - 27546450

AN - SCOPUS:84984856953

SN - 1046-5928

VL - 128

SP - 101

EP - 108

JO - Protein Expression and Purification

JF - Protein Expression and Purification

ER -

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

Abstract

Keywords

ASJC Scopus subject areas

Access to Document

Other files and links

Fingerprint

Cite this