Quantification of influenza virus mini viral RNAs using Cas13.

Abstract

Influenza A virus (IAV) RNA synthesis produces full-length and deletion-containing RNA molecules, which include defective viral genomes (DVG) and mini viral RNAs (mvRNA). Sequencing approaches have shown that DVG and mvRNA species may be present during infection, and that they can vary in size, segment origin, and sequence. Moreover, a subset of aberrant RNA molecules can bind and activate host pathogen receptor retinoic acid-inducible gene I (RIG-I), leading to innate immune signaling and the expression of type I and III interferons. Measuring the kinetics and distribution of these immunostimulatory aberrant RNA sequences is important for understanding their function in IAV infection. Here, we explored if IAV mvRNA molecules can be detected and quantified using amplification-free, CRISPR-LbuCas13a-based detection. We show that CRISPR-LbuCas13a can be used to measure the copy numbers of specific mvRNAs in samples from infected tissue culture cells. However, to efficiently detect mvRNAs in other samples, promiscuous CRISPR guide RNAs are required that activate LbuCas13a in the presence of multiple mvRNA sequences. One crRNA was able to detect full-length IAV segment 5 without amplification, allowing it to be used for general IAV infection detection nasopharyngeal swabs. Using CRISPR-LbuCas13a, we confirm that mvRNAs are present in ferret upper and lower respiratory tract tissue, as well as clinical nasopharyngeal swab extracts of hospitalized patients. Overall, CRISPR-LbuCas13a-based RNA detection is a useful tool for studying deletion-containing viral RNAs and it complements existing amplification-based approaches.