Genome-wide CRISPR/Cas9 screening to identify cell-specific response regulators to double-stranded RNA stress

Abstract

Double-stranded RNAs (dsRNAs) are key players in the innate immune response to viral infection. Upon infection, dsRNAs generated during viral transcription or the dsRNA viral genome itself are recognized by pattern recognition receptors (PRRs) on host cells, such as Toll-like receptors (TLRs), RIG-I-like receptors (RLRs), and cytosolic dsRNA sensors. This recognition leads to the activation of the downstream antiviral signalling pathways, including the production of type I interferons (IFNs) and pro-inflammatory cytokines and the induction of antiviral genes. In addition, human cells naturally encode cellular dsRNAs that can also activate innate immune response systems and drive pathogenesis of human diseases. The importance of dsRNA-mediated immune activation has led to the identification of various downstream regulators of the dsRNA stress response, such as PAMP receptors and dsRNA-binding proteins. However, an unbiased systematic approach to determine the key regulators and elucidate their regulatory mechanisms for dsRNAs is currently lacking. Moreover, despite the diverse cell-type signature and stress regulatory mechanism, there is a lack of cell-specific studies to understand dsRNA stress responses. Hence, we perform cell-specific genome-wide CRISPR/Cas9 knockout screening using poly(I:C) as an exemplary dsRNA stressor. Our screening hits in A549 validated previously known dsRNA receptors and regulators of inflammatory response. In neuronal progenitor cells (NPCs), our screening yielded interesting and distinct results, highlighting the cell-specific characteristics of dsRNA stress responses. Furthermore, to identify novel regulators, we compared our screening results with a list of putative dsRNA-binding proteins (dsRBPs). We identified the presence of novel proteins, including LSM12, NUFIP2, NOP16, and ATXN2L, which may play essential roles in regulating cell-specific dsRNA-mediated stress responses.