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CRISPR/Cas9-based editing of a sensitive transcriptional regulatory element to achieve cell type-specific knockdown of the NEMO scaffold protein
The use of alternative promoters for the cell type-specific expression of a given mRNA/protein is a common cell strategy. NEMO is a scaffold protein required for canonical NF-κB signaling. Transcription of the NEMO gene is primarily controlled by two promoters: one (promoter B) drives NEMO transcrip...
Autores principales: | , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2019
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6760803/ https://www.ncbi.nlm.nih.gov/pubmed/31553754 http://dx.doi.org/10.1371/journal.pone.0222588 |
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author | Babaei, Milad Liu, Yuekun Wuerzberger-Davis, Shelly M. McCaslin, Ethan Z. DiRusso, Christopher J. Yeo, Alan T. Kagermazova, Larisa Miyamoto, Shigeki Gilmore, Thomas D. |
author_facet | Babaei, Milad Liu, Yuekun Wuerzberger-Davis, Shelly M. McCaslin, Ethan Z. DiRusso, Christopher J. Yeo, Alan T. Kagermazova, Larisa Miyamoto, Shigeki Gilmore, Thomas D. |
author_sort | Babaei, Milad |
collection | PubMed |
description | The use of alternative promoters for the cell type-specific expression of a given mRNA/protein is a common cell strategy. NEMO is a scaffold protein required for canonical NF-κB signaling. Transcription of the NEMO gene is primarily controlled by two promoters: one (promoter B) drives NEMO transcription in most cell types and the second (promoter D) is largely responsible for NEMO transcription in liver cells. Herein, we have used a CRISPR/Cas9-based approach to disrupt a core sequence element of promoter B, and this genetic editing essentially eliminates expression of NEMO mRNA and protein in 293T human kidney cells. By cell subcloning, we have isolated targeted 293T cell lines that express no detectable NEMO protein, have defined genomic alterations at promoter B, and do not support activation of canonical NF-κB signaling in response to treatment with tumor necrosis factor. Nevertheless, non-canonical NF-κB signaling is intact in these NEMO-deficient cells. Expression of ectopic wild-type NEMO, but not certain human NEMO disease mutants, in the edited cells restores downstream NF-κB signaling in response to tumor necrosis factor. Targeting of the promoter B element does not substantially reduce NEMO expression (from promoter D) in the human SNU-423 liver cancer cell line. Thus, we have created a strategy for selectively eliminating cell type-specific expression from an alternative promoter and have generated 293T cell lines with a functional knockout of NEMO. The implications of these findings for further studies and for therapeutic approaches to target canonical NF-κB signaling are discussed. |
format | Online Article Text |
id | pubmed-6760803 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-67608032019-10-04 CRISPR/Cas9-based editing of a sensitive transcriptional regulatory element to achieve cell type-specific knockdown of the NEMO scaffold protein Babaei, Milad Liu, Yuekun Wuerzberger-Davis, Shelly M. McCaslin, Ethan Z. DiRusso, Christopher J. Yeo, Alan T. Kagermazova, Larisa Miyamoto, Shigeki Gilmore, Thomas D. PLoS One Research Article The use of alternative promoters for the cell type-specific expression of a given mRNA/protein is a common cell strategy. NEMO is a scaffold protein required for canonical NF-κB signaling. Transcription of the NEMO gene is primarily controlled by two promoters: one (promoter B) drives NEMO transcription in most cell types and the second (promoter D) is largely responsible for NEMO transcription in liver cells. Herein, we have used a CRISPR/Cas9-based approach to disrupt a core sequence element of promoter B, and this genetic editing essentially eliminates expression of NEMO mRNA and protein in 293T human kidney cells. By cell subcloning, we have isolated targeted 293T cell lines that express no detectable NEMO protein, have defined genomic alterations at promoter B, and do not support activation of canonical NF-κB signaling in response to treatment with tumor necrosis factor. Nevertheless, non-canonical NF-κB signaling is intact in these NEMO-deficient cells. Expression of ectopic wild-type NEMO, but not certain human NEMO disease mutants, in the edited cells restores downstream NF-κB signaling in response to tumor necrosis factor. Targeting of the promoter B element does not substantially reduce NEMO expression (from promoter D) in the human SNU-423 liver cancer cell line. Thus, we have created a strategy for selectively eliminating cell type-specific expression from an alternative promoter and have generated 293T cell lines with a functional knockout of NEMO. The implications of these findings for further studies and for therapeutic approaches to target canonical NF-κB signaling are discussed. Public Library of Science 2019-09-25 /pmc/articles/PMC6760803/ /pubmed/31553754 http://dx.doi.org/10.1371/journal.pone.0222588 Text en © 2019 Babaei et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. |
spellingShingle | Research Article Babaei, Milad Liu, Yuekun Wuerzberger-Davis, Shelly M. McCaslin, Ethan Z. DiRusso, Christopher J. Yeo, Alan T. Kagermazova, Larisa Miyamoto, Shigeki Gilmore, Thomas D. CRISPR/Cas9-based editing of a sensitive transcriptional regulatory element to achieve cell type-specific knockdown of the NEMO scaffold protein |
title | CRISPR/Cas9-based editing of a sensitive transcriptional regulatory element to achieve cell type-specific knockdown of the NEMO scaffold protein |
title_full | CRISPR/Cas9-based editing of a sensitive transcriptional regulatory element to achieve cell type-specific knockdown of the NEMO scaffold protein |
title_fullStr | CRISPR/Cas9-based editing of a sensitive transcriptional regulatory element to achieve cell type-specific knockdown of the NEMO scaffold protein |
title_full_unstemmed | CRISPR/Cas9-based editing of a sensitive transcriptional regulatory element to achieve cell type-specific knockdown of the NEMO scaffold protein |
title_short | CRISPR/Cas9-based editing of a sensitive transcriptional regulatory element to achieve cell type-specific knockdown of the NEMO scaffold protein |
title_sort | crispr/cas9-based editing of a sensitive transcriptional regulatory element to achieve cell type-specific knockdown of the nemo scaffold protein |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6760803/ https://www.ncbi.nlm.nih.gov/pubmed/31553754 http://dx.doi.org/10.1371/journal.pone.0222588 |
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