Table 1.
Clade-representing CCHFV sequences.
Table 2.
Primers, crRNAs and chemically synthesized CCHFV RNA fragments.
Table 3.
CCHFV-related viruses (Family Bunyaviridae).
Fig 1.
The CRISPR/Cas13a-based diagnostic assay (SHERLOCK) for CCHFV detection.
The polarity of an RNA strand is labelled for positive sense (+) or negative sense (-). In the RNA reporter, “F” and “Q” mean fluorophore and quencher, respectively.
Fig 2.
Design and function test of CCHFV CRISPR sets.
A and B. Sequences and genomic locations targeted by CCHFV CRISPR sets. The S segment sequences of CCHFV strains, labelled with GenBank accession numbers, were aligned at the assay design regions for CRISPR set 1 (in Panel A) and CRISPR set 2 (in Panel B). Nucleotides differing from the majority are highlighted in dark shades. Sequences and locations targeted by RT-RPA primers and crRNAs are indicated, based on the positive-sense sequence of the S segment. The CCHFV strains, representing each of all the seven clades/sub-clades, are detailed in Table 1, except that a Clade V variant, DQ211643, with a two-nucleotide deletion at genomic positions 31 and 32 is included only in this figure. Note that degenerate nucleotides were introduced into the crRNA of CRISPR set 2, to cover variations at each position in the targets, and are displayed with the standard codes by International Union of Pure and Applied Chemistry (IUPAC). C and D. Specific signal amplification by CRISPR Set 2, but not CRISPR set 1. The CRISPR sets were tested in the CRISPR/Cas13a-based SHERLOCK assay, in the presence of chemically synthesized CCHFV RNA fragments and different concentrations of stock crRNAs. Sequences of RT-RPA primers, crRNAs and synthetic RNA templates are listed in Table 2. Amplification plots show relative fluorescent units (RFU) at indicated time points in the T7-Cas13a reaction. C. Test of CRISPR set 1 (with constant crRNA spacer). PC RNA (positive control RNA, specific target) = Target_RNA_1–120; NC RNA (negative control RNA, non-specific target) = Target RNA_641–723. NTC (no-template control) = H2O. Graph represents five independent experiments showing similar patterns. D. Test of CRISPR set 2 (with degenerate crRNA spacer). PC RNA = Target RNA_641–723; NC RNA = Target_RNA_1–120. NTC = H2O. Graph represents four independent experiments showing similar patterns.
Fig 3.
The degenerate sequence-based CRISPR diagnostic detects CCHFV in a sensitive, specific and rapid manner.
A–G. Detection of all CCHFV clades/sub-clades. The S segment genomic RNAs representing different CCHFV clades/sub-clades were produced by in vitro transcription, serially diluted and tested for detection by the degenerate CRISPR set (CRISPR set 2). Amplification plots show relative fluorescent units (RFU) at indicated time points in the T7-Cas13a reaction. RNA template concentrations in the T7-Cas13a reaction are shown as copies/μl (cp/μl). Red curves: CCHFV RNA-containing samples; green solid curve (0 cp/μl): NTC (no-template control, H2O); and green dash curve (Neg): the negative threshold = Mean + 3X standard deviation of NTCs, calculated based on 12 NTC replicates. Any RFU value above the negative threshold is considered CCHFV positive. Graphs represent three independent experiments showing similar patterns. H. Limit of detection (LoD). The S segment genomic RNA from the Hoti strain was produced by in vitro transcription and tested at the indicated concentrations for detection by the degenerate CRISPR set. Positivity% values are shown based on 10 replicates per target concentration type. I. The degenerate sequence-based CRISPR diagnostic is specific for CCHFV, with no cross-reactivity against closely related viruses. RNAs from CCHFV and related viruses were tested for detection by the degenerate CRISPR set. NSD: Nairobi sheep disease virus; RVFV: Rift Valley fever virus; and NTC: no-template control (H2O). CCHFV and Rift Valley fever virus RNAs were extracted from cell culture-derived viruses. Other viral RNAs were the S segment genomic RNAs produced by in vitro transcription. RNA template concentrations in the T7-Cas13a reaction were all 40 pg/μl. Amplification plots show relative fluorescent units (RFU) at indicated time points in the T7-Cas13a reaction and represent three independent experiments showing similar patterns.
Fig 4.
The degenerate sequence-based CRISPR diagnostic is capable of detecting emerging CCHFV variants with new and more complex mutations.
Chemically synthesized CCHFV RNA targets were tested for detection at 1 cp/μl by the degenerate sequence-based CRISPR set, using crRNA_669–696 (crRNA_Deg), as in Fig 3. A constant sequence-based crRNA, derived from the Hoti strain, crRNA_669–696_5.Hoti (crRNA_Con), was compared to the degenerate sequence-based crRNA in the same context. The targeted CCHFV variants were isolates from India (IND, 2018) and United Arab Emirates (UAE, 2017) in Panels A and B, respectively, as well as a recombinant (Rec) between Clades I and IV. Sequences of synthetic RNA templates, RT-RPA primers and crRNAs and GenBank accession numbers of CCHFV strains are provided in Table 2. Sequence of the IND, UAE or Rec variant targeted by CRISPR was each aligned with the degenerate and constant spacer sequences of the crRNAs (all as CCHFV sense sequences). Each nucleotide in the crRNA spacers that differs from that in the targeted CCHFV variant is shaded. In crRNA_Deg, the region is underlined where each degenerate nucleotide covers its counterpart in the target sequence, meaning that one of the mixed nucleotide types represented by the degenerate code matches the counterpart nucleotide in the target. Arrow indicates the nucleotide in crRNA_Deg that does not cover/match the counterpart nucleotide in the target. In Panel C, regions in the target sequence with mutations characteristic of Clade I and Clade II are demarcated, respectively. Each amplification plot shows relative fluorescent units (RFU) at indicated time points in the T7-Cas13a reaction, in the presence (+) or absence (-) of the target CCHFV RNA, using an indicated crRNA. Data are represented as mean of three technical replicates.