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. 2023 Jun 28;93:104682. doi: 10.1016/j.ebiom.2023.104682

Fig. 1.

Fig. 1

CRISPR-CasRx gRNAs design strategy generates gRNAs with potent and specific antiviral activity in cells. a. Schematic of bioinformatics pipeline for gRNA prediction and scoring, as applied to enterovirus genomes. b. Visualization of scores and gRNAs against 795 enterovirus target genomic sequences. Top: Entropy Score (red) for each nucleotide of aligned viral genomes. Percentage of Conservation (blue) for each nucleotide among aligned viral genomes. Bottom: gRNAs on-target efficacy score along the target enterovirus, color-coded and divided into four quartiles. c. Designed gRNAs against the enterovirus RNA genome. Blue arrows: gRNAs with high scores. Red arrows: gRNAs with medium/low scores. d. Assessment of gRNA pools. 10K RD cells were seeded in each well of a 96-well plate and transduced with indicated AAVs. After 72 h, the cells were subjected to EV-A71 infection at MOI of 1 for 12 h, and the supernatant was then harvested for virus plaque assay (two-way ANOVA, p < 0.0001 for MOI, gRNAs, and MOIxgRNA interaction; Dunnett’s against eGFPg2). e. Assessment gRNAs against the 3D gene. 10K RD cells were seeded in each well of a 96-well plate and transduced with indicated AAVs. After 72 h, the cells were subjected to EV-A71 infection at MOI of 1 for 12 h, and the supernatant was then harvested for virus plaque assay (two-way ANOVA, p < 0.0001 for MOI, gRNAs, and MOIxgRNA interaction; Dunnett’s against eGFPg2). f. Multiplexed CasRx gRNAs on a single 3D target inhibits EV-A71 replication. RD cells were treated with AAV-CasRx bearing gRNAs at MOI of 1000K and infected with EV-A71 at MOI of 1 at 72 h later (one-way ANOVA, Dunnett’s against eGFPg2). g. Multiplexed gRNA pools inhibit EV-A71 plaque formation (all at a total MOI of 1000K) (two-way ANOVA, Dunnett’s against “0” eGFPg2). h. Specificity of CasRx-EV71_3Dguides pool in transduced human RD cells at MOI of 1000 K (n = 3 for both groups (EV-A71 gRNAs + EV-A71 infected) and (EV-A71 infected)). ∗adj-p < 0.05, ∗∗adj-p < 0.01, ∗∗∗adj-p < 0.005, ∗∗∗∗adj-p < 0.001.