Rapid and accurate detection of novel coronavirus SARS-CoV-2 using CRISPR-Cas3

Abstract

Novel coronavirus SARS-CoV-2 outbreaks have rapidly spread to multiple countries, highlighting the urgent necessity for fast, sensitive, and specific diagnostic tools for virus surveillance. Here, the previously unknown collateral single-stranded DNA cleavage we observed with type I CRISPR-Cas3 highlights its potential for development as a Cas3-mediated rapid (within 40 min), low-cost, instrument-free detection method for SARS-CoV-2. This Cas3-based assay is comparable with Cas12- and real-time reverse-transcriptase PCR-based assays in its speed and sensitivity, but offers greater specificity for single-base-pair discrimination while negating the need for highly trained operators. These findings support the use of CRISPR diagnostics for point-of-care testing in patients with suspected SARS-CoV-2 infections.

Dynamic mechanisms of CRISPR interference by Escherichia coli CRISPR-Cas3

“…To quantitatively measure collateral ssDNA cleavage activity we used a FQ-labeled untargeted ssDNA probe 40,41 (Fig. 1e), which is used in CRISPR-based diagnostics as a platform for rapid and sensitive nucleic acid detection, for example in Covid-19 test kits [43][44][45] .…”

“…To analyze DNA cleavage activity, 1.6 nM of plasmid with or without targeted sequences were added to 115 nM EcoCascade-crRNA complex, 250 nM EcoCas3, and 2.5 mM ATP in CRISPR-Cas3 working buffer (60 mM KCl, 10 mM MgCl 2 , 10 µM CoCl 2 , 5 mM HEPES-KOH, pH 7.5), as previously described 40,41,45 . After incubation at 37°C, samples were detected by either electrophoresis or with the MultiNa microchip electrophoresis system and the DNA-12,000 kit (Shimadzu, Kyoto, Japan).…”

Intrinsic Signal Amplification by Type-III CRISPR-Cas Systems Provides a Sequence-Specific Viral Diagnostic

“…The LoD of type III CRISPR-based fluorometric detection (Fig. 1C) is roughly 100-10,000 fold better than direct RNA or DNA detection by Cas12, Cas13 or Cascade (Supplemental Table 1) (Chen et al, 2018;Gootenberg et al, 2018;Yoshimi et al, 2020). However, it is currently not sensitive enough to directly detect SARS-CoV-2 in all patients capable of spreading the infection, which requires detecting 10 6 RNA copies/mL (La Scola et al, 2020;Larremore et al, 2020;Paltiel et al, 2020;Wölfel et al, 2020).…”

Viral Related Tools against SARS-CoV-2

“…This CRISPR-based tool employs mainly Cas3 endonuclease, in combination with Cas5, 6, 7, 8, and 11, which mediates targeted DNA cleavage. When combined with isothermal amplification methods, CONAN provides a rapid and sensitive method to detect SARS-CoV-2, with a reliability of 90% [ 119 ]. CRISPR-COVID: A few months ago, another CRISPR-based tool suitable for the diagnostic of SARS-CoV-2 infection was developed, also based on the Cas13a endonuclease.…”

“…This CRISPR-based tool employs mainly Cas3 endonuclease, in combination with Cas5, 6, 7, 8, and 11, which mediates targeted DNA cleavage. When combined with isothermal amplification methods, CONAN provides a rapid and sensitive method to detect SARS-CoV-2, with a reliability of 90% [ 119 ].…”