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

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


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).

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • CRISPR-Cas Systems
  • Cells, Cultured
  • Cyclooxygenase 2 / genetics
  • Gene Knockout Techniques
  • Genes, Reporter
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • Humans
  • Immunity, Innate / genetics
  • Macrophages / immunology
  • Macrophages / metabolism*
  • Mice
  • NF-kappa B / genetics*
  • NF-kappa B / metabolism*
  • RNA, Long Noncoding / genetics*
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Signal Transduction


  • NF-kappa B
  • RNA, Long Noncoding
  • Recombinant Fusion Proteins
  • Green Fluorescent Proteins
  • Ptgs2 protein, mouse
  • Cyclooxygenase 2