Characterization of H1N1 and interferon-β-responsive long non-coding RNAs using single-cell CRISPRi in human lung epithelial and immune cell lines 2260688

Abstract Introduction Despite 36,000 long non-coding RNAs (lncRNAs) annotated in the human genome, functional characterization remains limited, and the roles of lncRNAs in the interferon (IFN) response are largely unknown. We previously identified VILMIR as an IFN-stimulated lncRNA that enhances the host IFN response. Through analysis of public bulk RNA-seq datasets, we identified two additional lncRNAs that are consistently upregulated following H1N1 influenza infection and interferon-β (IFN-β) treatment. This study aims to identify immune genes that are perturbed by candidate lncRNA knockdowns (KDs) and explore the relationship between KD efficiency of lncRNA-targeting guide RNAs and the scale of transcriptomic perturbations. Methods To investigate the roles of these lncRNAs, we used CRISPRi to generate stable KD cell lines in human lung epithelial cells, monocytes, and T-lymphoblasts. These cell lines were treated with IFN-β for 6 hours, and mock versus IFN-treated samples were analyzed using 10x CRISPRi scRNA-seq analysis. Results Transcriptome analysis of each lncRNA KD line compared to controls revealed a set of differentially expressed immune genes in response to IFN-β stimulation. Conclusion In summary, we have identified two novel interferon-responsive human lncRNAs that may play important roles in the regulation of the host interferon response across different cell types. The results provide mechanistic targets for future studies and serve as preliminary data for future large-scale screens of candidate lncRNAs. Funding Source National Institutes of Health Grant R21AI147187 Topic Categories Immune Response Regulation: Molecular Mechanisms (IRM)