Fig. 3: Genome-wide CRISPR knockout (KO) screen with a stable chikungunya virus (CHIKV) replicon cell line.

a Schematic of CHIKV replicon used in this study. SP6, engineered promoter for in vitro transcription; sg CHIKV sub-genomic promoter, UTR untranslated region, nsP nonstructural protein, eGFP enhanced green fluorescent protein (green)-Zeocin drug resistance (light blue) reporter-selection gene cassette. b Host factor knockouts (KOs) in the CHIKV replicon cells. CHIKV replicon activity was quantified as change in % eGFP reporter expression (in ≥1000 cells) in wild-type (WT) CHIKV replicon cells (black symbols), CHIKV replicon cells with KO of G3BP1, a host gene established to be involved in replication, or KO of MXRA8 a host gene required for CHIKV entry (open symbol and gray symbol, respectively). Values represent mean % eGFP ± standard deviation (SD) for technical triplicates, normalized to % eGFP at the start of the experiment. c CRISPR KO screen gene enrichment. X-axis = genes; y-axis = enrichment scores calculated by MAGeCK robust rank aggregation (RRA) analysis of genes detected in the sorted (eGFP-low) versus unsorted (control) cell populations; colors highlight biological pathways among candidate hits. d Validation of replicon screen phenotype. eGFP fluorescence 18 d after parental (WT) CHIKV-2 replicon cells were treated with non-targeting (NT) control guide RNA (gRNA) or individual gRNAs targeting candidate gene hits. Values represent mean % eGFP ± SD normalized to wild-type (WT) control (3 biological replicates; n ≥ 1000 cells). Asterisks denote P-values (*p = 0.03, **p = 0.002, and ***p < 0.001) for one-way ANOVA with Dunnett’s multiple comparisons test. e RT-qPCR analysis of CHIKV infections in KO cells. Independent KO cell populations in the Huh7.5.1-Cas9 (WT) background generated with gRNAs targeting gene hits from screen (open symbols) or non-targeting gRNAs (NT, black symbol) were incubated with CHIKV LR-2006 OPY1 (multiplicity of infection [MOI] = 0.1) and harvested after 7, 18, and 25 h. Values for each timepoint represent the mean ± SD of technical triplicates of normalized CHIKV RNA expression levels relative to the normalized levels detected in the NT cells. Gene names are abbreviated according to the human standard (https://www.genenames.org/). Source data are provided as a Source data file.