Immunity and Viral Infections: Modulating Antiviral Response via CRISPR–Cas Systems

Figures

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  Figure 1. Sensing of foreign nucleic acids. (A) TLR- and RLR-mediated sensing of foreign nucleic acids. Different types of cytoplasmic foreign RNA are recognized by RIG-I or MDA5 sensors followed by activation of MAVS and downstream TBK1-IRF signaling. In endosomes, RNA and CpG-DNA activate TLRs that result in one of the two signaling pathways involving TRIF or MYD88-IRAK. Activation of IRFs induces the expression of interferons and mRNA of pro-inflammatory factors. (B) Cytoplasmic and nuclear sensors of foreign DNA. Cytoplasmic DNA can be sensed by a number of sensors, including IFI16, cGAS, and AIM2. The first two factors activate the STING pathway that ultimately induces TBK1/IRF and interferon secretion. Upon recognition of cytoplasmic DNA, AIM2 induces caspase-1-dependent maturation of pro-IL-1β (pro-inflammatory response). A2B1 (hnRNPA2B1) and IFI16, among other factors, can participate in the sensing of foreign DNA in the nuclei of cells. IFI16 interferes with foreign nuclear DNA by epigenetic silencing, whereas A2B1 recognizes foreign DNA as well as activates and enhances innate antiviral responses. This picture was created in BioRender. Abbreviation: A2B1—hnRNPA2B1;…

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  Figure 2. Restriction of viral life cycle by different ISGs for viruses with nuclear replication. ISGs with antiviral activity are shown for different steps of viral replication, including binding and viral entry, capsid disassembly, nuclear import, reverse transcription, viral nucleic acid replication/transcription, nuclear export, translation, capsid assembly, budding and release of viral particles. This picture was created in BioRender.

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  Figure 3. Mechanisms of viral immune evasion. (A) Evasion of immune recognition. (B) Blockade of interferon signaling. This picture was created in BioRender. Abbreviations: NA—nucleic acids; ISG—interferon-stimulated genes; IFNs—interferons; PTM—post-transcriptional modifications; SOCS—suppressor of cytokine signaling.

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  Figure 4. General pipeline of pooled CRISPR screens. (1) In silico design of sgRNA targeting regulatory elements (promoters and/or enhancers) of target genes (or in genome-wide format). In modern pooled libraries, four to 10 different sgRNAs are designed to target a single regulatory element, which constitutes up to 200,000 sgRNAs in a single genome-wide library. (2) Lentiviral delivery of CRISPR pooled library into cells expressing appropriate Cas protein (Cas9 for knockout screens and dCas9 for activation of interference screens) into infected cells at low MOI (to ensure delivery of a single sgRNA into a single cell). (3) Selection of transduced cells (negative or positive). Note that knockdown of genes (KO) is relevant for nucleolytic Cas systems and base editors, whereas CRISPRa and CRISPRi systems cause the overexpression or suppression of gene transcription, correspondingly. (4) Enrichment of cells. (5) Isolation of nucleic acids from the bulk of enriched cells. (6) Deep sequencing and identification of hits by determining sgRNAs representation in the material. This picture was created in BioRender.