Abrogation of PRRSV infectivity by CRISPR-Cas13b-mediated viral RNA cleavage in mammalian cells

Abstract

CRISPR/Cas9 enables dsDNA viral genome engineering. However, the lack of RNA targeting activities limits the ability of CRISPR/Cas9 to combat RNA viruses. The recently identified class II type VI CRISPR/Cas effectors (Cas13) are RNA-targeting CRISPR enzymes that enable RNA cleavage in mammalian and plant cells. We sought to knockdown the viral RNA of porcine reproductive and respiratory syndrome virus (PRRSV) directly by exploiting the CRISPR/Cas13b system. Effective mRNA cleavage by CRISPR/Cas13b-mediated CRISPR RNA (crRNA) targeting the ORF5 and ORF7 genes of PRRSV was observed. To address the need for uniform delivery of the Cas13b protein and crRNAs, an all-in-one system expressing Cas13b and duplexed crRNA cassettes was developed. Delivery of a single vector carrying double crRNAs enabled the simultaneous knockdown of two PRRSV genes. Transgenic MARC-145 cells stably expressing the Cas13b effector and crRNA mediated by lentiviral-based transduction showed a robust ability to splice the PRRSV genomic RNA and subgenomic RNAs; viral infection was almost completely abrogated by the combination of double crRNAs simultaneously targeting the ORF5 and ORF7 genes. Our study indicated that the CRISPR/Cas13b system can effectively knockdown the PRRSV genome in vitro and can potentially be used as a potent therapeutic antiviral strategy.

Figure 1

Characterization of the CRISPR/Cas13b system in PRRSV mRNA targeting. (a) Schematic diagram of the design of crRNAs targeting the ORF7 mRNA transcript, template strand of the ORF7 gene and CMV promoter. (b) Schematic diagram showing the steps of the determination of the effect of CRISPR/Cas13b on PRRSV gene knockdown by co-transfection of the three plasmids into HEK293T cells. (c) Microscopic fluorescence images showing the expression of the PRRSV ORF7-eGFP reporter after CRISPR/Cas13b activity with various targeting crRNAs. The bar indicates 100 μm. (d,e) PRRSV N protein expression and ORF7 mRNA levels were determined by flow cytometry and quantitative RT-PCR, respectively. Values shown as the mean ± SEM with n = 3. *and **refer to P values < 0.05 and 0.01, respectively.

Figure 2

Determination of the most potent crRNA for Cas13b-mediated PRRSV ORF5 and ORF7 targeting. (a) Locations of crRNA targeting regions within the ORF5 and ORF7 genes. (b) RNA cleavage efficiency was determined for the indicated crRNAs targeting ORF5 and ORF7 by qRT-PCR and normalized to the non-targeting (NT) control.

Figure 3

Development of an all-in-one CRISPR/Cas13b-duplexed crRNA delivery system. (a) The structure of the all-in-one delivery vector and PRRSV gene reporter. The U6 promoter drives duplexed guide expression, and the EF1a promoter mediates the transcription of Cas13b. The eGFP reporter fused with Cas13b by a 2 A self-cleaving peptide facilitates the detection of Cas13b expression. (b,c,d) The all-in-one Cas13b system carrying specific PRRSV ORF5 crRNA significantly cleaved ORF5 mRNA. However, no accumulative effect of Cas13b was detected by targeting the ORF5 gene with two crRNAs simultaneously (b,c), flow cytometry; (d), real-time PCR). (e) The all-in-one plasmid was further modified to incorporate triple crRNAs targeting the ORF7 gene. Representative flow cytometry graphs for eGFP- and RFP657-positive cells are shown. (f) The reduction percentages of dual-marker positive cells were determined by flow cytometry. Each crRNA group was normalized to the NT control. (g) The percentage of RNA reduction was determined by qRT-PCR. (h) The established platform enables the simultaneous knockdown of PRRSV ORF5 and ORF7 mRNA by incorporating two corresponding guide protospacer sequences. Values are shown as mean ± SEM with n = 3. **, ***and NS refer to P values < 0.01, 0.001 and no significant differences, respectively.

Figure 4

Cas13b mediates the efficient knockdown of the PRRSV genome in lentiviral transgenic MARC-145 cells. (a) Schematic diagram for lentiviral transfer gene constructs encoding Cas13b and crRNAs. (b) Determination of Cas13 effector expression levels in puro-selected transgenic cells by flow cytometry analysis. (c) The expression of corresponding crRNAs in each cell line was detected by PCR. PCR products were separated by 5% agarose gels. (d) The growth kinetics of HP-PRRSV strain 10PL01 with an MOI of 0.1 in transgenic cells. (e) The Cas13b cleavage activity on PRRSV genomic RNA was determined by qRT-PCR with primers targeting the NSP9 gene. (f) The PRRSV subgenomic RNA levels were measured by qRT-PCR with a set of specific primers targeting each subgenomic RNA. (g) Comparison of each subgenomic RNA knockdown efficiency between cells expressing Cas13b-crRNA 5-2 and Cas13b-crRNA 7-1. Values are shown as the mean ± SEM with n = 3. ***refers to P value < 0.001.