Researchers developed a new detection assay for SARS-CoV-2.
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Genetic Engineering Publications - GEG Tech top picks
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Fluorescence in situ hybridization (FISH) is a powerful molecular technique for detecting nucleic acids in cells. However, it requires cell fixation and denaturation. Wang et al. found that CRISPR-Cas9 protects guide RNAs from degradation in cells only when bound to target DNA. Taking advantage of this target-dependent stability switch, they developed a labeling technique, named CRISPR LiveFISH, to detect DNA and RNA using fluorophore-conjugated guide RNAs with Cas9 and Cas13, respectively. CRISPR LiveFISH improves the signal-to-noise ratio, is compatible with living cells, and allows tracking real-time dynamics of genome editing, chromosome translocation, and transcription.
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In a recent study published in Proceedings of the National Academy of Sciences, researchers developed a new assay for the detection of SARS-CoV-2. For the test, CRISPR systems were coupled to loop-mediated isothermal amplification (LAMP) or recombinase polymerase (RPA) amplification to boost sensitivity and improve detection at attomolar levels. In addition, reverse transcription (RT)-RPA/LAMP systems coupled with Cas13 systems have been developed for the detection of SARS-CoV-2. RT-LAMP has become the preferred method due to its high sensitivity, low cost and ease of use. Since these systems use functional Cas enzymes at 37°C and LAMP requires high temperatures (55°C to 65°C), the RT-LAMP/RPA assays coupled with CRISPR. The multiplexed assay therefore has a thermophilic Cas13a isolated from Thermoclostridium caenicola (TccCas13a) and uses it for the detection of SARS-CoV-2 using fluorescent amidite-labelled RNA and has developed an AapCas12b-based RNase P detection using HEX-labelled ssDNA reporters. The researchers achieved simultaneous detection of RNase P and SARS-CoV-2 without any interference from the fluorescence signals. A mobile phone application was developed to collect and interpret the fluorescence viewer readings at low cost.