Degenerate sequence-based CRISPR diagnostic for Crimean–Congo hemorrhagic fever virus
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.