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Harnessing CRISPR-Cas13a Towards the Direct Detection of RNA Viruses

  • Author(s): Fozouni, Parinaz
  • Advisor(s): Roan, Nadia
  • et al.
Abstract

Viral diagnostics largely rely on reverse transcription before PCR-based amplification of the viral genome, which cannot be easily performed outside a specialized laboratory. There is an urgent need for rapid and portable diagnostics that can quantitatively detect viruses. The December 2019 outbreak of a novel respiratory virus, SARS-CoV-2, has become an ongoing global pandemic due in part to the challenge of quickly identifying and isolating asymptomatic and pre-symptomatic carriers of the virus. CRISPR diagnostics can augment gold-standard PCR-based testing if they can be made rapid, portable and accurate. Here, we report the development of an amplification-free CRISPR-Cas13a assay for the direct detection and quantification of viral RNA using a mobile phone microscope. When applied to the detection of SARS-CoV-2 RNA, the assay achieved ∼100 copies/μL sensitivity in under 30 minutes of measurement time and accurately detected pre-extracted RNA from a set of positive clinical samples in under 5 minutes. To improve sensitivity and specificity, we combined crRNAs targeting SARS-CoV-2 RNA, and directly quantified viral load using enzyme kinetics. Integrated with a reader device based on a mobile phone, this assay has the potential to enable rapid, low-cost, point-of-care screening for SARS-CoV-2. We further applied this approach towards the direct detection of HIV-1 RNA, which could serve as a tool for both identifying HIV at the earliest stage of initial infection and detecting HIV rebound in treated individuals following treatment interruption. Together, this work lays the framework for the development of amplification-free CRISPR diagnostics towards RNA viruses.

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