Deployable CRISPR-Cas13a diagnostic tools to detect and report Ebola and Lassa virus cases in real-time

Abstract

Recent outbreaks of viral hemorrhagic fevers (VHFs), including Ebola virus disease (EVD) and Lassa fever (LF), highlight the urgent need for sensitive, deployable tests to diagnose these devastating human diseases. Here we develop CRISPR-Cas13a-based (SHERLOCK) diagnostics targeting Ebola virus (EBOV) and Lassa virus (LASV), with both fluorescent and lateral flow readouts. We demonstrate on laboratory and clinical samples the sensitivity of these assays and the capacity of the SHERLOCK platform to handle virus-specific diagnostic challenges. We perform safety testing to demonstrate the efficacy of our HUDSON protocol in heat-inactivating VHF viruses before SHERLOCK testing, eliminating the need for an extraction. We develop a user-friendly protocol and mobile application (HandLens) to report results, facilitating SHERLOCK's use in endemic regions. Finally, we successfully deploy our tests in Sierra Leone and Nigeria in response to recent outbreaks.

Designing viral diagnostics with model-based optimization

“…ADAPT leverages diverse genome data and predictive models to design optimal nucleic acid diagnostic assays for viruses. Early versions of ADAPT have previously been applied to detecting Lassa virus 51 and to specific detection of 169 human viruses and influenza subtyping 40 . We also used ADAPT to design a SARS-CoV-2 assay in January, 2020, as well as assays for 66 related viruses 52 ; though designed from only the 20 genomes available at the time 53 , we predict the SARS-CoV-2 assay to detect 99.8% of the ~180,000 genomes available in mid-November, 2020.…”

“…Though designed from only the 20 genomes available at the time 50 , we predict the SARS-CoV-2 assay to detect 99.8% of the ~180,000 genomes available in mid-November, 2020. Primitive versions of ADAPT were also applied to design CRISPR-based assays for detecting 169 human viruses and influenza subtyping 37 , and CRISPR-based diagnostics for Lassa virus 51 .…”

“…For these two models, which can capture and extract spatial relationships in the input, we used an alternative input (labeled 'One-hot (2D)' in figures). Here, the input dimensionality is (48,8) and consists of a concatenated one-hot encoding of the target and guide sequence. Namely, each element x i (i ∈ {1 .…”

CRISPR/Cas technology as a promising weapon to combat viral infections

“…The fluorophore is then released, resulting in an increase in fluorescence (Figure 4). Both sensors have been used for the detection of several viruses with aM sensitivity [ 103,108–111 ] (Table 3), including SARS‐CoV‐2. [ 108,111 ] In fact, in May 2020 the FDA authorized the use of SHERLOCK for COVID‐19 detection, and it has recently been clinically validated.…”

“…have recently developed a mobile phone application that enables the quantification of the viral load from the paper strip based on the band intensity. [ 110 ]…”