Colorimetric Detection of SARS-CoV-2 and Drug-Resistant pH1N1 Using CRISPR/dCas9

Viruses have been a continuous threat to human beings. The coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to a pandemic that is still ongoing worldwide. Previous pandemic influenza A virus (pH1N1) might be re-emerging through a drug-resistant mutation. We report a colorimetric viral detection method based on the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 endonuclease dead (dCas9) system. In this method, RNA in the viral lysate was directly recognized by the CRISPR/dCas9 system with biotin-protospacer adjacent motif (PAM)-presenting oligonucleotide (PAMmer). Streptavidin-horseradish peroxidase then bound to biotin-PAMmer, inducing a color change through the oxidation of 3,3',5,5'-tetramethylbenzidine. Using the developed method, we successfully identified SARS-CoV-2, pH1N1, and pH1N1/H275Y viruses by the naked eye. Moreover, the detection of viruses in human nasopharyngeal aspirates and sputum was demonstrated. Finally, clinical samples from COVID-19 patients led to a successful diagnosis. We anticipate that the current method can be employed for simple and accurate diagnosis of viruses.