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dc.contributor.authorBabaei, Miladen_US
dc.contributor.authorLiu, Yuekunen_US
dc.contributor.authorWuerzberger-Davis, Shelly M.en_US
dc.contributor.authorMcCaslin, Ethan Z.en_US
dc.contributor.authorDiRusso, Christopher J.en_US
dc.contributor.authorYeo, Alan T.en_US
dc.contributor.authorKagermazova, Larisaen_US
dc.contributor.authorMiyamoto, Shigekien_US
dc.contributor.authorGilmore, Thomas D.en_US
dc.date.accessioned2020-04-15T15:11:35Z
dc.date.available2020-04-15T15:11:35Z
dc.identifier.citationMilad Babaei, Yuekun Liu, Shelly M Wuerzberger-Davis, Ethan Z McCaslin, Christopher J DiRusso, Alan T Yeo, Larisa Kagermazova, Shigeki Miyamoto, Thomas D Gilmore. "CRISPR/Cas9-based editing of a sensitive transcriptional regulatory element to achieve cell type-specific knockdown of the NEMO scaffold protein." PLOS ONE, Volume 14, Issue 9. https://doi.org/10.1371/journal.pone.0222588
dc.identifier.issn1932-6203
dc.identifier.urihttps://hdl.handle.net/2144/40177
dc.description.abstractThe 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, noncanonical NF-κB signaling is intact in these NEMO-deficient cells. Expression of ectopic wildtype 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 SNU423 liver cancer cell line. Thus, we have created a strategy for selectively eliminating cell typespecific 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.en_US
dc.languageen
dc.language.isoen_US
dc.publisherPublic Library of Science (PLOS)en_US
dc.relation.ispartofPLOS ONE
dc.rightsCopyright: © 2019 Babaei et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.en_US
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/
dc.subjectGeneral science & technologyen_US
dc.titleCRISPR/Cas9-based editing of a sensitive transcriptional regulatory element to achieve cell type-specific knockdown of the NEMO scaffold proteinen_US
dc.typeArticleen_US
dc.description.versionPublished versionen_US
dc.identifier.doi10.1371/journal.pone.0222588
pubs.elements-sourcecrossrefen_US
pubs.notesEmbargo: Not knownen_US
pubs.organisational-groupBoston Universityen_US
pubs.organisational-groupBoston University, College of Arts & Sciencesen_US
pubs.organisational-groupBoston University, College of Arts & Sciences, Department of Biologyen_US
pubs.publication-statusPublished onlineen_US
dc.date.online2019-09-25
dc.identifier.mycv506578


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Copyright: © 2019 Babaei et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Except where otherwise noted, this item's license is described as Copyright: © 2019 Babaei et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.