Background:
Coxsackievirus B (CVB) is a positive-sense, single-stranded RNA virus and one of the major etiological agents of viral myocarditis. As CVB replicates exclusively through its RNA genome, direct targeting and degradation of viral RNA represents a promising therapeutic approach. The CRISPR–Cas13 system, an RNA-guided RNA-targeting nuclease, offers a powerful platform to achieve specific RNA cleavage without altering the host genome.
Aim:
We aimed to establish and evaluate an optimized hfCas13X–crRNA system for inhibiting CVB3 replication in cardiomyocytes and to explore its potential as an antiviral gene therapy for CVB-associated myocarditis.
Methods and Results:
Based on our previous development of a highly efficient hfCas13X-based all-in-one gene-silencing construct, we generated a novel hfCas13X-based system with improved specificity and compact size, suitable for AAV packaging.
Using in silico design via the “Chop-Chop” platform, 25 crRNAs targeting conserved CVB genome regions were cloned individually into pAAV-CMV-hfCas13X-U6-crRNA vectors and verified by Sanger sequencing. To pre-screen crRNA efficiency, luciferase reporter constructs containing the respective viral fragments (Pmir-F1–F5) were established. Co-transfection assays in HEK293 cells identified crRNA “gRNA1” as the most potent guide, achieving significant reduction in luciferase signal compared to controls.
Although hfCas13X showed slightly reduced cleavage efficiency compared to Cas13d, it exhibited superior specificity and reduced collateral activity, favorable for translational applications.
For delivery optimization, four AAV serotypes (AAV2/6/9, MyoAAV) were tested in HL-1 mouse cardiomyocytes expressing EGFP reporters. AAV6 achieved the highest transduction efficiency (≈85.9%), outperforming other serotypes, and was selected for downstream studies.
In HL-1 cells transduced with AAV6-hfCas13X-crRNA1, subsequent CVB3 infection (MOI 0.1) led to a marked suppression of viral replication, as quantified by TCID50 assays in Vero cells. Preliminary results indicate that hfCas13X–crRNA1 significantly inhibits viral propagation, confirming the antiviral potential of this system. Validation in human iPSC-derived cardiomyocytes is ongoing.
Conclusion:
We demonstrate that the hfCas13X-crRNA system effectively targets and suppresses CVB3 replication in cardiomyocytes. The optimized AAV6-mediated delivery provides a robust platform for future in vivo applications. This study establishes a promising foundation for RNA-targeted gene therapy against CVB-associated viral myocarditis.
