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Published on: August 10, 2018
AAV-CRISPR-Cas13 eliminates human enterovirus and prevents death of infected mice
Choong Tat Keng1, Thinesshwary Yogarajah2, Regina Ching Hua Lee3
1Genome Institute of Singapore, Agency for Science, Technology and Research, 60 Biopolis Street, Singapore 138672, Singapore.
Background:
RNA viruses account for many human diseases and pandemic events but are often not targetable by traditional therapeutics modalities. Here, we demonstrate that adeno-associated virus (AAV) -delivered CRISPR-Cas13 directly targets and eliminates the positive-strand EV-A71 RNA virus in cells and infected mice.
Methods:
We developed a Cas13gRNAtor bioinformatics pipeline to design CRISPR guide RNAs (gRNAs) that cleave conserved viral sequences across the virus phylogeny and developed an AAV-CRISPR-Cas13 therapeutics using in vitro viral plaque assay and in vivo EV-A71 lethally-infected mouse model.
Findings:
We show that treatment with a pool of AAV-CRISPR-Cas13-gRNAs designed using the bioinformatics pipeline effectively blocks viral replication and reduces viral titers in cells by >99.99%. We further demonstrate that AAV-CRISPR-Cas13-gRNAs prophylactically and therapeutically inhibited viral replication in infected mouse tissues and prevented death in a lethally challenged EV-A71-infected mouse model.
Interpretation:
Our results show that the bioinformatics pipeline designs efficient CRISPR-Cas13 gRNAs for direct viral RNA targeting to reduce viral loads. Additionally, this new antiviral AAV-CRISPR-Cas13 modality represents an effective direct-acting prophylactic and therapeutic agent against lethal RNA viral infections.
Funding:
Agency for Science, Technology and Research (A∗STAR) Assured Research Budget, A∗STAR Central Research Fund UIBR SC18/21-1089UI, A∗STAR Industrial Alignment Fund Pre-Positioning (IAF-PP) grant H17/01/a0/012, MOE Tier 2 2017 (MOE2017-T2-1-078; MOE-T2EP30221-0005), and NUHSRO/2020/050/RO5+5/NUHS-COVID/4.
Insights
This study introduces adeno-associated virus (AAV)-delivered CRISPR-Cas13 as a novel antiviral therapy. This CRISPR-Cas13 system effectively targets and eliminates RNA viruses, offering a new prophylactic and therapeutic strategy against lethal viral infections.
Area of Science:
- Molecular Biology
- Virology
- Gene Editing
Background:
- RNA viruses cause significant human diseases and pandemics.
- Traditional therapeutics often struggle to target these viruses effectively.
- Adeno-associated virus (AAV)-delivered CRISPR-Cas13 offers a novel approach to directly target viral RNA.
Purpose of the Study:
- To develop and evaluate an AAV-delivered CRISPR-Cas13 system for targeting and eliminating the positive-strand EV-A71 RNA virus.
- To assess the efficacy of this system in cell cultures and a mouse model of lethal viral infection.
Main Methods:
- Developed a Cas13gRNAtor bioinformatics pipeline for designing CRISPR guide RNAs (gRNAs) targeting conserved viral sequences.
- Constructed AAV-CRISPR-Cas13 therapeutics.
- Evaluated efficacy using in vitro viral plaque assays and an in vivo EV-A71 lethally-infected mouse model.
Main Results:
- A pool of AAV-CRISPR-Cas13-gRNAs significantly reduced viral replication and titers in cells by over 99.99%.
- The AAV-CRISPR-Cas13 system demonstrated prophylactic and therapeutic inhibition of viral replication in mouse tissues.
- Treatment prevented mortality in a lethal EV-A71-infected mouse model.
Conclusions:
- The bioinformatics pipeline successfully designs efficient CRISPR-Cas13 gRNAs for direct viral RNA targeting.
- AAV-CRISPR-Cas13 is a potent antiviral modality for reducing viral loads.
- This approach serves as an effective direct-acting prophylactic and therapeutic agent against lethal RNA viral infections.
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