AAV-CRISPR-Cas13 eliminates human enterovirus and prevents death of infected mice

Figures

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  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.…

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  Fig. 2. Pan-enteroviral inhibition by AAV2-CasRx-EV71_3Dguides. a. Assessment of inhibitory activity of the six-gRNAs pool targeting HFM41 strain on three other EV71 variants (H strain, B5 strain, and C4 strain). 10K RD cells were transduced with AAV2-CasRx at MOI of 10K, 100K, and 1000K bearing either GFP guide2 or EV-A71 guide(s) for 72 h and then subjected to EV-A71 infection at MOI of 1 for 12 h, after which the supernatant is harvested for virus plaque assay. b. The pool of 6 gRNAs designed against the HFM41 strain exhibits antiviral inhibition against three other EV71 variants (H strain, B5 strain, and C4 strain) at an MOI of 1000K. c. Each pool of 6 gRNAs is engineered specifically for the sequences of the H, B5, or C4 strain. d. Each re-engineered pool of 6 gRNAs exhibits antiviral inhibition against their respective H, B5, or C4 strain. In all panels, comparison between two groups (CasRx-GFPg2 and CasRx-EV71_3Dguides) was analyzed by Student t-test (two-tailed). ∗p < 0.05, ∗∗p < 0.005, ∗∗∗p < 0.001.

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  Fig. 3. AAVDJ-CRISPR-CasRx confers prophylactic protection in a mouse model for hand, foot and mouth disease. a. BALB/c mice were injected intraperitoneally with a dose of 1 × 1011 vgs or 1 × 1012 vgs of AAVDJ-EV71gRNAs per mouse at 2 days old and subsequently injected intraperitoneally with EV-A71 at a dose of 2 × 107 PFU per mouse at 5 days old. 1 × 1012 vgs AAVDJ-GFPgRNA was used as control. b. Survival curve of treated mice over 19 days post-infection (dpi). c. The body weight of each mouse in each treatment group over 19 dpi. Comparison between two groups (CasRx-GFPg2 and CasRx-EV71_3Dguides) was analyzed by Log-rank (Mantel–Cox) test. ∗p < 0.05, ∗∗p < 0.005. d. The clinical score of mice was recorded using the mice clinical assessment scoring system (M-CASS) involving observation of five markers: activity, breathing, movement, body weight, and dehydration over 19 dpi. e. Treatment reduces virus titers in the hind limbs of mice as determined using viral plaque assay. Viral titration results were from a single experiment with multiple mice. f. Treatment reduces virus titers in the brain of mice as determined using viral plaque assay. Comparison between two groups (CasRx-GFPg2 and…

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  Fig. 4. AAVDJ-CRISPR-CasRx prevents mortality and reduces pathology when applied 24 h after EV-A71 infection. a. BALB/c mice were injected intraperitoneally with a dose of 2 × 107 PFU EV-A71 per mouse at 5 days old. After 24 h, mice were injected intraperitoneally with a dose of 1 × 1011 viral genomes (vgs) or 1 × 1012 vgs of AAVDJ-EV71gRNAs per mouse. 1 × 1012 vgs AAVDJ-GFPgRNA was used as treatment control. b. The survival of the mice was recorded for 19 days post-infection (dpi). Comparison between two groups (CasRx-GFPg2 and CasRx-EV71_3Dguides) was analyzed by Log-rank (Mantel–Cox) test. ∗p < 0.05. c. The body weight of each mouse in each treatment group was recorded for 19 dpi. d. The clinical score of each mouse was recorded using the mice clinical assessment scoring system (M-CASS). e. Virus titers in the hind limbs of mice from different treatment groups were determined using the plaque-forming assay. Viral titration results were from a single experiment with multiple mice. f. Virus titers in the brains of mice from different treatment groups were determined using the plaque-forming assay. Comparison between two groups (CasRx-GFPg2 and CasRx-EV71_3Dguides) was analyzed by…