Gene sequencing in back of reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is the current approach for discriminating infections produced by different severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants in the clinic. However, sequencing is often a time-consuming step, which hinders the deployment of a very fast response during a pandemic. Here, we propose to run a CRISPR-Cas12a reaction after completing the RT-qPCR and in the very same pot to detect with high specificity genetic marks characterizing variants of concern. A crRNA was appropriately designed to detect the S gene of the SARS-CoV-2 Omicron BA.1 variant. A significant response with >20-fold dynamic range was obtained for the Omicron BA.1 S gene, while the Delta S gene did not produce any detectable signal. The sensitivity of the method was analyzed with a series of diluted samples and different Cas12a nucleases. A correlation between the RT-qPCR CT values and the CRISPR-Cas12a reaction signals was observed. Variant discrimination with the CRISPR-Cas12a reaction was possible in some minutes with high accuracy from patient samples. In conclusion, CRISPR-Cas systems seem ready to be exploited in the clinic to boost personalized diagnoses and accelerate epidemiological surveillance in a cost-effective way.
© 2024 The Authors. Published by American Chemical Society.