Metagenomic sequencing of zoonotic viruses: evaluation of a CRISPR-Cas–based rRNA depletion system
DOI:
https://doi.org/10.12834/VetIt.3908.38985.2Keywords:
CRISPR-Cas, metagenomics, NGS, rRNA, virusAbstract
Pathogen-agnostic diagnostics are crucial for the early detection of emerging viruses. Shotgun metagenomic sequencing enables unbiased detection of viral genomes but is frequently constrained by the abundance of host and microbial ribosomal RNA (rRNA), which reduces sensitivity and increases sequencing costs. CRISPR-Cas9–based rRNA depletion has emerged as an alternative to enzymatic methods; however, its performance for the characterization of zoonotic viruses across diverse animal hosts and tissues remains underexplored. We compared CRISPR-Cas9 (Jumpcode CRISPRclean™ Plus) and RNase H–based enzymatic depletion (Ribo-Zero Plus, Illumina) using 12 samples positive for rabies lyssavirus, influenza A virus, West Nile virus or norovirus, from multiple host species and tissues, including both high-quality and degraded RNA. CRISPR-Cas9 efficiently reduced rRNA content (14.5%) but recovered fewer viral reads than Ribo-Zero, which achieved up to 60.7× enrichment. Both methods produced complete viral consensus genomes when RNA quality and viral load were sufficient. However, based on the data generated here, enzymatic depletion currently remains more efficient and cost-effective for viral metagenomics. Further optimization of CRISPR-Cas9 workflows could enhance its utility for viral surveillance and diagnostics.References
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