Field-deployable viral diagnostics using CRISPR-Cas13

Abstract

Mitigating global infectious disease requires diagnostic tools that are sensitive, specific, and rapidly field deployable. In this study, we demonstrate that the Cas13-based SHERLOCK (specific high-sensitivity enzymatic reporter unlocking) platform can detect Zika virus (ZIKV) and dengue virus (DENV) in patient samples at concentrations as low as 1 copy per microliter. We developed HUDSON (heating unextracted diagnostic samples to obliterate nucleases), a protocol that pairs with SHERLOCK for viral detection directly from bodily fluids, enabling instrument-free DENV detection directly from patient samples in <2 hours. We further demonstrate that SHERLOCK can distinguish the four DENV serotypes, as well as region-specific strains of ZIKV from the 2015-2016 pandemic. Finally, we report the rapid (<1 week) design and testing of instrument-free assays to detect clinically relevant viral single-nucleotide polymorphisms.

Fig. 1.. ZIKV and DENV detection from patient samples and clinical isolates.

(A) Schematic of SHERLOCK. Nucleic acid is extracted from clinical samples, and the target is amplified by RPA using either RNA or DNA as input (RT-RPA or RPA, respectively). RPA products are detected in a reaction containing T7 RNA polymerase, Cas13, a target-specific crRNA, and an RNA reporter that fluoresces when cleaved. We tested SHERLOCK on (B) cDNA derived from 37 patient samples collected during the 2015–2016 ZIKV pandemic and (C) cDNA from 16 patient samples that were compared head-to-head with the Altona RealStar Zika Virus RT-PCR assay. +: ZIKV seed stock cDNA (3×10² cp/μl) and ×: no input. (D) SHERLOCK on RNA extracted from 24 DENV-positive patient samples and clinical isolates. Dashed blue line: threshold for presence or absence of ZIKV or DENV (see Methods).

Fig. 2.. Direct detection of ZIKV and DENV in bodily fluids with HUDSON and SHERLOCK.

(A) Schematic of direct viral detection using HUDSON and SHERLOCK. (B-C) Detection of ZIKV RNA in particles diluted in healthy human serum (pink, B) or healthy human saliva (blue, C). Same PBS control used in A and B as experiments were performed together. (D) Detection of ZIKV RNA in particles diluted in healthy human urine (yellow). Black: non-diluted viral stock. Grey: dilutions in PBS. Error bars indicate 1 S.D. based on 3 technical replicates. (E-F) Detection of DENV RNA directly from patient serum (E) and saliva samples (F). (G) Lateral flow detection of DENV from samples shown in (E-F). All samples were treated with TCEP/EDTA prior to heating.

Fig. 3.. Multivirus panels can differentiate viral species and serotypes.

(A-C) Panel of 4 related flaviviruses (A) used to detect individual viral targets (B) or paired viral targets (C) using species-specific crRNAs with 3 hours of detection. (D-E) Identification of DENV serotypes 1–4 using serotype-specific crRNAs (D), tested using synthetic targets with 3 hours of detection (E). (F) Identification of DENV serotypes in extracted RNA from patient samples. Each row is a sample, each column is a crRNA, target-specific fluorescent values are normalized by row. Purple: DENV serotype identified. Synthetic targets were used at 10⁴ cp/μl. Error bars indicate 1 S.D. based on 3 technical replicates. We expect off-target crRNAs to have close to zero target-specific fluorescence (see Methods). Primer, crRNA, and target sequences are in Tables S5–7.

Fig. 4.. Identification of adaptive and functional ZIKV mutations.

(A) SHERLOCK assays for SNP identification. Dark colors: derived crRNAs, light colors: ancestral crRNAs. (B-C) Three region-specific SNPs from the 2015–2016 ZIKV pandemic, including genomic locations of the SNPs and a simplified phylogenetic tree, tested using synthetic targets (10⁴ cp/μl) and viral seedstock cDNA (3×10² cp/μl). (D-E) Identification of region-specific SNPs in ZIKV cDNA samples from the Dominican Republic (DOM), United States (USA), and Honduras (HND). The fluorescence ratio (derived crRNA fluorescence divided by ancestral crRNA fluorescence) for each SNP in each sample is shown in a bar plot (log₂-transformed data in a heatmap). (F) Timeline for developing a SHERLOCK assay for a new SNP (more detail in Fig. S26). (G-H) Identification of a microcephaly-associated ZIKV mutation (PrM S139N) by fluorescent and colorimetric detection. In all panels, error bars indicate 1 S.D. based on 3 technical replicates.