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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.

ABSTRACT: 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-?B 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-?B signaling in response to treatment with tumor necrosis factor. Nevertheless, non-canonical NF-?B 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-?B 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-?B signaling are discussed.

SUBMITTER: Babaei M

PROVIDER: S-EPMC6760803 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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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.

Babaei Milad M Liu Yuekun Y Wuerzberger-Davis Shelly M SM McCaslin Ethan Z EZ DiRusso Christopher J CJ Yeo Alan T AT Kagermazova Larisa L Miyamoto Shigeki S Gilmore Thomas D TD

PloS one 20190925 9

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 o ...[more]

PMID: 31553754

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Project description:The discovery and exploitation of the prokaryotic adaptive immunity system based on clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins have revolutionized genetic engineering. CRISPR-Cas tools have enabled extensive genome editing as well as efficient modulation of the transcriptional program in a multitude of organisms. Progress in the development of genetic engineering tools for the genus Clostridium has lagged behind that of many other prokaryotes, presenting the CRISPR-Cas technology an opportunity to resolve a long-existing issue. Here, we applied the Streptococcus pyogenes type II CRISPR-Cas9 (SpCRISPR-Cas9) system for genome editing in Clostridium acetobutylicum DSM792. We further explored the utility of the SpCRISPR-Cas9 machinery for gene-specific transcriptional repression. For proof-of-concept demonstration, a plasmid-encoded fluorescent protein gene was used for transcriptional repression in C. acetobutylicum Subsequently, we targeted the carbon catabolite repression (CCR) system of C. acetobutylicum through transcriptional repression of the hprK gene encoding HPr kinase/phosphorylase, leading to the coutilization of glucose and xylose, which are two abundant carbon sources from lignocellulosic feedstocks. Similar approaches based on SpCRISPR-Cas9 for genome editing and transcriptional repression were also demonstrated in Clostridium pasteurianum ATCC 6013. As such, this work lays a foundation for the derivation of clostridial strains for industrial purposes.ImportanceAfter recognizing the industrial potential of Clostridium for decades, methods for the genetic manipulation of these anaerobic bacteria are still underdeveloped. This study reports the implementation of CRISPR-Cas technology for genome editing and transcriptional regulation in Clostridium acetobutylicum, which is arguably the most common industrial clostridial strain. The developed genetic tools enable simpler, more reliable, and more extensive derivation of C. acetobutylicum mutant strains for industrial purposes. Similar approaches were also demonstrated in Clostridium pasteurianum, another clostridial strain that is capable of utilizing glycerol as the carbon source for butanol fermentation, and therefore can be arguably applied in other clostridial strains.

Dataset Information

CRISPR/Cas9-based editing of a sensitive transcriptional regulatory element to achieve cell type-specific knockdown of the NEMO scaffold protein.

Publications

CRISPR/Cas9-based editing of a sensitive transcriptional regulatory element to achieve cell type-specific knockdown of the NEMO scaffold protein.

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