Infectious Diseases - Research Publications

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Type: Preprint × Author: Lewin, Sharon ×

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    Reprogrammed CRISPR-Cas13b suppresses SARS-CoV-2 replication and circumvents its mutational escape through mismatch tolerance
    Fareh, M ; Zhao, W ; Hu, W ; Casan, JML ; Kumar, A ; Symons, J ; Voskoboinik, I ; Ekert, P ; Rudraraju, R ; Lewin, S ; Trapani, J ( 2020)

    ABSTRACT

    Mutation-driven evolution of SARS coronavirus-2 (SARS-CoV-2) highlights the need for innovative approaches that simultaneously suppress viral replication and circumvent viral escape routes from host immunity and antiviral therapeutics. Here, we employed genome-wide computational prediction and singlenucleotide resolution screening to reprogram CRISPR-Cas13b against SARS-CoV-2 genomic and subgenomic RNAs. Reprogrammed Cas13b effectors targeting accessible regions of Spike and Nucleocapsid transcripts achieved >98% silencing efficiency in virus free-models. Further, optimized and multiplexed gRNAs suppressed viral replication by up to 90% in mammalian cells infected with replication-competent SARS-CoV-2. Unexpectedly, the comprehensive mutagenesis of guide-target interaction demonstrated that single-nucleotide mismatches do not impair the capacity of a potent single gRNA to simultaneously suppress ancestral and mutated SARS-CoV-2 in infected mammalian cells, including the highly infectious and globally disseminated Spike D614G mutant. The specificity, efficiency and rapid deployment properties of reprogrammed Cas13b described here provide a molecular blueprint of antiviral therapeutics to simultaneously suppress a wide range of SARS-CoV-2 mutants, and is readily adaptable to other emerging pathogenic viruses.
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    Evaluation of serological tests for SARS-CoV-2: Implications for serology testing in a low-prevalence setting
    Bond, K ; Nicholson, S ; Ming Lim, S ; Karapanagiotidis, T ; Williams, E ; Johnson, D ; Hoang, T ; Sia, C ; Purcell, D ; R Lewin, S ; Catton, M ; P Howden, B ; A Williamson, D ( 2020)

    Background

    Robust serological assays are essential for long-term control of the COVID-19 pandemic. Many recently released point-of-care (PoCT) serological assays have been distributed with little pre-market validation.

    Methods

    Performance characteristics for five PoCT lateral flow devices approved for use in Australia were compared to a commercial enzyme immunoassay (ELISA) and a recently described novel surrogate virus neutralisation test (sVNT).

    Results

    Sensitivities for PoCT ranged from 51.8% (95% CI 43.1 to 60.4%) to 67.9% (95% CI 59.4–75.6%), and specificities from 95.6% (95% CI 89.2–98.8%) to 100.0% (95% CI 96.1–100.0%). Overall ELISA sensitivity for either IgA or IgG detection was 67.9% (95% CI 59.4–75.6), increasing to 93.8% (95% CI 85.0–98.3%) for samples > 14 days post symptom onset. Overall, sVNT sensitivity was 60.9% (95% CI 53.2–68.4%), rising to 91.2%% (95% CI 81.8–96.7%) for samples collected > 14 days post-symptom onset, with a specificity 94.4% (95% CI 89.2–97.5%),

    Conclusion

    Performance characteristics for COVID-19 serological assays were generally lower than those reported by manufacturers. Timing of specimen collection relative to onset of illness or infection is crucial in the reporting of performance characteristics for COVID-19 serological assays. The optimal algorithm for implementing serological testing for COVID-19 remains to be determined, particularly in low-prevalence settings.