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

ABSTRACT: The innate immune system protects against infections by initiating an inducible inflammatory response. NF-?B 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-?B expression is unknown. Here, we describe the generation of a GFP-based NF-?B 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-?B reporter line, enabling us to screen for genes that regulate NF-?B signaling. Our deletion-based approach identified two long intergenic non-coding(linc)RNAs, lincRNA-Cox2 and lincRNA-AK170409, that control NF-?B signaling. We demonstrate a potential novel role for lincRNA-Cox2 in promoting I?B? 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-?B-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-?B regulators, and this tool will be useful for identifying additional genes involved in regulating this transcription factor critical for immune function.

SUBMITTER: Covarrubias S 

PROVIDER: S-EPMC5743067 | biostudies-literature | 2017 Dec

REPOSITORIES: biostudies-literature