Journal: bioRxiv
Article Title: Mitochondrial dsRNA: A Hidden Source of Immunogenic RNA in ADAR1 Deficiency
doi: 10.64898/2026.01.06.698045
Figure Lengend Snippet: ( A ) Workflow of the phenotypic CRISPR screening. GFP fluorescence was measured by flow cytometry to quantify the level of immune response activation and cells with the highest (top 2%; ‘IFN-high’) and lowest (bottom 5%; ‘IFN-low’) GFP signals were sorted and collected for sgRNA sequencing. MAGeCK bioinformatic pipeline was used to identify potential negative regulators of IFN response. Two independent screens were performed. Created in BioRender. Shen, H. (2025) https://BioRender.com/a69p174 . ( B ) Volcano plots illustrating the top hits identified from the CRISPR screen conducted in ADAR1 KO HEK293T cells. Each dot represents a gene, with x -axis displaying the log 2 (Fold change) calculated based on the enrichment of sgRNAs targeting the indicated gene in ‘IFN-high’ population versus ‘IFN-low’ population, and y -axis showing the -log 10 Robust Rank Aggregation (RRA) score. Significance was determined using thresholds of FDR < 0.05 and |log 2 (Fold change)| > 1, as indicated by dot size. Genes significantly enriched in the ‘IFN-high’ population are highlighted as red dots. Mitochondrial protein-coding genes (mito-genes) and previously reported innate immune repressors are listed in the upper-left and upper-right corners, respectively. The numbers preceding the gene names represent their respective rankings. ( C ) Gene ontology (GO) analysis of genes enriched in the ‘IFN-high’ population in ADAR1 KO cells. Dot size corresponds to the number of genes with each GO category, while the x -axis and dot color indicate the -log 10 (p-value), reflecting the statistical significance of the enrichment. ( D ) Quantitative PCR (qPCR) analysis of expression changes in identified mito-genes and IFNB1 , along with western blotting (WB) of GFP and ADAR1 expression levels, following treatment with siRNAs targeting the indicated mito-genes in both NT and ADAR1 KO HEK293T cells. Two siRNAs were used for each target gene. Each dot represents the mean value of technical triplicates from an independent experiment. Data are presented as the mean ± S.D. of 3 independent experiments (biological replicates). siNC, non-targeting siRNA.
Article Snippet: Extracted RNA was reverse-transcribed using Advantage RT-for PCR kit (Clontech) with random hexamer followed by qPCR.
Techniques: CRISPR, Fluorescence, Flow Cytometry, Activation Assay, Sequencing, Real-time Polymerase Chain Reaction, Expressing, Western Blot
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