Protection of animals against devastating RNA viruses using CRISPR-Cas13s

Figure 1. Schematic workflow of the used approach in this study to inhibit FMDV infection using the CRISPR-Cas system (1) Transmission and circulation of FMD disease between wildlife-livestock ecosystems due to animal interactions. (2) Finding conserved regions among different FMDV serotypes by considering multiple criteria and designing efficient and specific crRNAs by the CaSilico tool. (3) Designed crRNAs of type VI-A/B/D were synthesized as DNA oligos, and then, each crRNA was cloned into separate crRNA cloning backbones. In addition to crRNA designing, several shRNAs were designed to compare virus knockdown by RNAi and CRISPR-Cas systems. (4) Virus propagation and titration of two FMDV serotypes (O and A) were performed on BHK-21 cells to be used for viral challenge. (5) The sgRNA screening was performed to validate potent crRNAs. (6) Two methods, RT-qPCR and TCID50 assay were used to measure viral RNA and FMDV infectivity, respectively. (7) CRISPR-Cas systems can potentially be leveraged to aid antiviral development for viral diseases in animals. Figure was created with BioRender.com.

Figure 2. Schematic of FMDV genome and targeted regions by CRISPR-Cas and RNAi systems Scissors indicate target sites. Conserved regions of the 3D gene were tiled with nine crRNAs belonging to different subtypes of CRISPR-Cas type VI. LwaCas13a, PspCas13b, and RfxCas13d spacers are indicated in blue, green, and orange, respectively. The nucleotides in red highlight residues in the guide (antisense) strand of shRNA. Proper non-targeting controls as negative control “scramble” for nonspecific effects were designed. NTCs have the shuffled nucleotide sequence of the target-specific crRNA or shRNA and have passed characteristics of a functional crRNA or shRNA such as off-target analysis (Table 1).

Figure 3. Effect of Cas13 and RNAi on FMDV inhibition, serotype O (A) Experimental procedure for evaluating Cas13s and RNAi antiviral activity in BHK-21 cells. BHK-21 cells were transfected with plasmids containing targeting and non-targeting shRNAs or Cas13 and crRNAs that target or do not target FMDV 24 h before infection. Then the supernatants were collected 24 hpi to evaluate the antiviral activity. Silencing efficiency of LwaCas13a (B), PspCas13b (C), RfxCas13d (D), and RNAi system (E) on FMDV virus according to RT-qPCR results. In (B) to (E), data points in the graph are five independent biological experiments performed in technical replicates, lines indicate means; error bars represent SD; t test was performed to compare each treatment to non-targeting crRNA or shRNA (green and red color of the boxes indicate p < 0.05 and p < 0.01, respectively).

Figure 4. Effect of Cas13 and RNAi on FMDV inhibition, serotype A Silencing efficiency of LwaCas13a (A), PspCas13b (B), and the RNAi system (C) on FMDV virus according to RT-qPCR results. In (A)–(C), data points in the graph are four or five independent biological experiments performed in technical replicates, lines indicate means; error bars represent SD; t test was performed to compare each treatment to non-targeting crRNA or shRNA (red color of the boxes indicates p < 0.01).

Figure 5. Effect of Cas13 and RNAi on FMDV infectivity (A) Experimental schematic for evaluation of infectious particles production inhibition. BHK-21 cells were co-transfected with LwaCas13a and L3D2 or LNTC crRNA encoding plasmids or plasmids encoding sh2C and SNTC shRNA 24 h before infection with FMDV serotype O. Then the supernatants were collected 24 hpi to quantify infectious virions using TCID50 assay. (B) Representative data of the TCID50 assay. The number of infectious virions was expressed as FMDV plaque-forming units/mL (PFU/mL). In (B), lines indicate means; error bars represent SD; t test was performed to compare the two treatments (red color of the bar indicates p < 0.01).