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

Jin Cui,Navapon Techakriengkrai,Sanipa Suradhat,Teerawut Nedumpun

doi:10.1038/s41598-020-66775-3

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.

Highlights

RNA viruses remain a great threat to humans and animals around the world[1,2]
We aimed to explore whether the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas13b system could directly knocking down Porcine reproductive and respiratory syndrome virus (PRRSV) mRNA
To assess whether the ORF7 mRNA could be repressed by CRISPR/Cas13b targeting, HEK293T cells were simultaneously co-transfected with Cas13b, ORF7-eGFP and individual CRISPR RNA (crRNA) (Fig. 1b)

Summary

Introduction

RNA viruses remain a great threat to humans and animals around the world[1,2]. Vaccination is the primary strategy to prevent viral infections in hosts[3]. In addition to RNA knockdown capability, fusion of catalytically inactive Cas[13] (dCas13) to an RNA-editing enzyme retained the RNA-binding activity and enabled specific RNA editing in mammalian cells[40] These advantages of type VI CRISPR systems have the potential to be developed as a platform to combat RNA viruses by targeting and degrading viral RNA in mammalian cells. The CRISPR/Cas13b system enabled an almost complete knockdown of PRRSV genomic and subgenomic RNAs to eradicate viral infections in lentiviral-mediated transgenic MARC-145 cells expressing Cas13b and duplexed crRNAs. Our study indicated the potential of using the CRISPR/Cas13b system as a novel therapeutic strategy by directly targeting the viral genes of RNA viruses

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Conclusion