Targeting pseudoknots with Cas13b inhibits porcine epidemic diarrhoea virus replication

Abstract

Clustered regularly interspaced short palindromic repeats-associated protein 13 (CRISPR-Cas13), an RNA editing technology, has shown potential in combating RNA viruses by degrading viral RNA within mammalian cells. In this study, we demonstrate the effective inhibition of porcine epidemic diarrhoea virus (PEDV) replication and spread using CRISPR-Cas13. We analysed the sequence similarity of the pseudoknot region between PEDV and severe acute respiratory syndrome coronavirus 2, both belonging to the Coronaviridae family, as well as the similarity of the RNA-dependent RNA polymerase (RdRp) gene region among three different strains of the PED virus. Based on this analysis, we synthesized three CRISPR RNAs (crRNAs) targeting the pseudoknot region and the nonpseudoknot region, each for comparison. In cells treated with crRNA #3 targeting the pseudoknot region, RdRp gene expression decreased by 95%, membrane (M) gene expression by 89% and infectious PEDV titre within the cells reduced by over 95%. Additionally, PED viral nucleocapsid (N) and M protein expression levels decreased by 83 and 98%, respectively. The optimal concentration for high antiviral efficacy without cytotoxicity was determined. Treating cells with 1.5 µg of Cas13b mRNA and 0.5 µg of crRNA resulted in no cytotoxicity while achieving over 95% inhibition of PEDV replication. The Cas13b mRNA therapeutics approach was validated as significantly more effective through a comparative study with merafloxacin, a drug targeting the pseudoknot region of the viral genome. Our results indicate that the pseudoknot region plays a crucial role in the degradation of the PEDV genome through the CRISPR-Cas13 system. Therefore, targeting Cas13b to the pseudoknot offers a promising new approach for treating coronavirus infections.

Keywords: CRISPR RNAs (crRNAs); CRISPR-Cas13b; RNA editing; porcine epidemic diarrhoea virus (PEDV); pseudoknot; viral replication.

Fig. 1.. Comparison of PEDV variant pseudoknot sequences and crRNA production. (a) Alignment of slippery and pseudoknot sequences between SARS-CoV-2 and PEDV. The alignment compares nucleotide sequences, highlighting the slippery site (boxed) and pseudoknot site (grey) between SARS-CoV-2 and PEDV. Identical nucleotides are marked with asterisks (*) and differences with periods (.) below the sequences. Gaps are shown as dashes (-). (b) Sequence diversity among PEDV variants, including CV777, OH851 and CKK1-1. The top diagram shows the general genome organization of PEDV. The bottom diagram shows the multiple sequence alignment generated via CLUSTALW that compares the slippery region (boxed) and pseudoknot region (grey) among three PEDV variants. (c) CRISPR-Cas13b system targeting the RdRp) gene of PEDV. The diagram illustrates CRISPR-Cas13b targeting the RdRp gene, indicating the pseudoknot and nonpseudoknot crRNA sites. (d) Schematic of the secondary structures of the PEDV slippery and pseudoknot regions with crRNA targeting sites marked. Sequences targeted by crRNAs #1, #2 and #3 are indicated by the red, green and blue bands, respectively.

Fig. 2.. Potent inhibition of PEDV transmission via pseudoknot-targeting crRNA #3. (a) Quantification of PEDV gene expression. Vero cells were transfected with Cas13b mRNA and crRNAs targeting CKK1-1 of the PEDV strain 24 h before infection. After 24 h, RNA was extracted, and PEDV RdRp and membrane gene expression levels were quantified using qRT-PCR. Results were normalized to RNA expression in cells transfected with nontargeting crRNAs. (b) Representative images of PEDV protein (green) and DAPI (blue) staining in Vero cells. Viral protein levels from (a) were visualized under a fluorescence microscope. Scale bar: 50 µm. (c) Quantification of the percentage of PEDV protein-positive cells relative to DAPI-positive cells, tested as in (b). Green bars represent PEDV protein-positive cells compared with total cells (DAPI-positive cells). (d) Total proteins from the infected cells in (a) were analysed using a western blot assay. Intensity ratios of PEDV proteins, normalized to GAPDH, are shown above and below each corresponding protein band. (e) Quantification of PEDV titre. Vero cells were infected with the virus from (a). Titration was performed using a plaque assay at 4 dpi. The dilution factor multiplied by the plaque count was used to determine the virus titre (n=3 for each group). Values are means±sem. One-way ANOVA was used for statistical analysis. ***P<0.0001.

Fig. 3.. Optimization of Cas13b and crRNA for enhanced antiviral activity against PEDV in a dose-dependent manner. (a) Quantification of PEDV gene copies. Vero cells were transfected with 0.5 µg of either crRNA #3 or nontarget crRNA and varying amounts of Cas13b mRNA (0.5, 1.0, 1.5 or 2.0 µg). At 24 h post-transfection, cells were infected with PEDV (0.05 m.o.i.). After another 24 h, PEDV RdRp and membrane gene expression levels in total RNA were quantified using qRT-PCR. Results were normalized to RNA expression in cells transfected with nontargeting crRNAs. (b) The same RNA transfection from (a) was followed by a luminescent cell viability assay (ATP assay) 48 h later. Bioluminescence values were normalized to those of untreated cells. (c) Vero cells were transfected with 2 µg of Cas13b mRNA and various amounts of crRNA #3 (0.1, 0.2, 0.5 or 1.0 µg). After 24 h, the cells were infected with PEDV, and 24 h later, PEDV RdRp and membrane gene expression levels were quantified using qRT-PCR. Results were normalized to RNA expression in cells transfected with nontargeting crRNAs. (d) Luminescent cell viability assay (ATP assay) was performed 48 h after transfection, as shown in (c). Bioluminescence values were normalized to those of untreated cells. Values are means±sem. One-way ANOVA was used for statistical analysis. ***P<0.0001.

Fig. 4.. Inhibition of PEDV transmission via merafloxacin, a pseudoknot formation inhibitor. (a) Quantification of PEDV gene copies. Vero cells were infected with PEDV and treated with the indicated merafloxacin concentrations 1 h later. After 24 h, PEDV RdRp gene expression was quantified using qRT-PCR. Results were normalized to RNA expression in cells treated with DMSO. (b) Representative immunofluorescence images of PEDV protein (green) and DAPI (blue) staining in Vero cells. Images are from one of three replicates. Scale bars: 50 µm. (c) Quantification of the percentage of PEDV protein-positive cells relative to DAPI-positive cells, tested as in (b). (d) Cytotoxicity quantification following merafloxacin treatment. Vero cells were treated with the indicated concentrations of merafloxacin dissolved in DMSO. After 24 h, a luminescent cell viability assay (ATP assay) was performed. Bioluminescence values were normalized to those of untreated cells. Values are means±sem.