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Journal of General Virology header logo Journal of General Virology

Volume 106, Issue 2

Targeting pseudoknots with Cas13b inhibits porcine epidemic diarrhoea virus replication Open Access

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 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, gene expression decreased by 95%, membrane () gene expression by 89% and infectious PEDV titre within the cells reduced by over 95%. Additionally, PED viral nucleocapsid () 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.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): CRISPR RNAs (crRNAs) , CRISPR-Cas13b , porcine epidemic diarrhoea virus (PEDV) , pseudoknot , RNA editing and viral replication
Funding
This study was supported by the:
  • Ministry of Education (Award 2017R1A6A1A03015876)
    • Principal Award Recipient: DongseobTark
  • Korea Health Industry Development Institute (Award HI22C1637)
    • Principal Award Recipient: SangminKang
© 2025 The Authors
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License.
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/content/journal/jgv/10.1099/jgv.0.002071
2025-02-04
2025-11-16

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Targeting pseudoknots with Cas13b inhibits porcine epidemic diarrhoea virus replication
J. Gen. Virol. 106, 002071 (2025); https://doi.org/10.1099/jgv.0.002071
/content/journal/jgv/10.1099/jgv.0.002071
/content/journal/jgv/10.1099/jgv.0.002071
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