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J Biol Chem. 2017 Dec 22;292(51):20911-20920. doi: 10.1074/jbc.M117.799155. Epub 2017 Oct 19.

CRISPR/Cas-based screening of long non-coding RNAs (lncRNAs) in macrophages with an NF-κB reporter.

Author information

1
From the Department of Molecular, Cell, and Developmental Biology and.
2
Center for Biomolecular Science and Engineering, University of California, Santa Cruz, California 95064 and.
3
the Department of Microbiology and Immunology, Diabetes Center, University of California San Francisco, San Francisco, California 94143.
4
From the Department of Molecular, Cell, and Developmental Biology and sucarpen@ucsc.edu.

Abstract

The innate immune system protects against infections by initiating an inducible inflammatory response. NF-κB is one of the critical transcription factors controlling this complex response, but some aspects of its regulation remain unclear. For example, although long non-coding RNAs (lncRNAs) have been shown to critically regulate gene expression, only a fraction of these have been functionally characterized, and the extent to which lncRNAs control NF-κB expression is unknown. Here, we describe the generation of a GFP-based NF-κB reporter system in immortalized murine bone marrow-derived macrophages (iBMDM). Activation of this reporter, using Toll-like receptor ligands, resulted in GFP expression, which could be monitored by flow cytometry. We also established a CRISPR/Cas9 gene deletion system in this NF-κB reporter line, enabling us to screen for genes that regulate NF-κB signaling. Our deletion-based approach identified two long intergenic non-coding(linc)RNAs, lincRNA-Cox2 and lincRNA-AK170409, that control NF-κB signaling. We demonstrate a potential novel role for lincRNA-Cox2 in promoting IκBα degradation in the cytoplasm. For lincRNA-AK170409, we provide evidence that this nuclearly-localized lincRNA regulates a number of inflammation-related genes. In conclusion, we have established an NF-κB-GFP iBMDM reporter cell line and a line that stably expresses Cas9. Our approach enabled the identification of lincRNA-Cox2 and lincRNA-AK170409 as NF-κB regulators, and this tool will be useful for identifying additional genes involved in regulating this transcription factor critical for immune function.

KEYWORDS:

CRISPR/Cas; NF-κB (NF-κB); inflammation; long non-coding RNA (long ncRNA, lncRNA); macrophage; toll-like receptor (TLR)

PMID:
29051223
PMCID:
PMC5743067
DOI:
10.1074/jbc.M117.799155
[Indexed for MEDLINE]
Free PMC Article

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