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.