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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.

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From the Department of Molecular, Cell, and Developmental Biology and.
Center for Biomolecular Science and Engineering, University of California, Santa Cruz, California 95064 and.
the Department of Microbiology and Immunology, Diabetes Center, University of California San Francisco, San Francisco, California 94143.
From the Department of Molecular, Cell, and Developmental Biology and


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.


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

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