CRISPR/Cas13a-assisted AMP generation for SARS-CoV-2 RNA detection using a personal glucose meter

-cleavage of CRISPR/Cas13a, generating 2',3'-cyclic phosphate adenosine, which is converted to adenosine monophosphate (AMP) by the T4 polynucleotide kinase. Subsequently, the AMP is converted to adenosine diphosphate (ADP) through phosphorylation by a myokinase; ADP is then used as a substrate in the cascade enzymatic reaction promoted by pyruvate kinase and hexokinase. The overall reaction leads to the continuous conversion of glucose to glucose-6-phosphate, resulting in a reduction of glucose concentration proportional to the level of target RNA, which can therefore be indirectly measured with a PGM. By employing this novel strategy, severe acute respiratory syndrome coronavirus-2 RNA can be successfully detected with excellent specificity. In addition, we were able to overcome non-specific responses of CRISPR/Cas13a and distinguish single nucleotide polymorphisms by introducing a single-base mismatch in the complementary RNA. Our study provides an alternative coronavirus disease 2019 detection technology that is affordable, accessible, and portable with a fast turnaround time and excellent selectivity.