Systematically Investigating CRISPR/Cas12a Fluorescent Biosensor for Sensitive and Specific Single Nucleotide Variants Detection

Precise identification and detection of single nucleotide variation (SNV) concomitant with excess wild-type DNA is greatly needed for invasive disease diagnosis, pathogens detection and early prediction of drug responsiveness. Many variants of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), notably the D614G and N501Y mutations, have been shown to significantly increase the infectivity of pandemics. We herein investigated CRISPR/Cas12a integrated three types fluorescent reporters and two crRNAs for SNV detection by taking D614G and N501Y variants of SARS-CoV-2 as model examples. We systematically screened all possible base substitutions from positions 0 to 19 and identified the middle position of crRNA could efficiently increase the specificity from both theoretical and experimental standpoints. With selected mutation location of crRNA, we then investigated the specificity of ssDNA, dsDNA and molecular beacon (MB) fluorescent reporters and proved the MB reporters can efficiently increase the discriminatory factors. Furthermore, we designed an additional mutation site on crRNA to increase the specificity. For user convenience, we engineered the lateral flow strips to present the results visualized with the naked eyes. Results of specific variants from Omicron proved the feasibility of clinical applications. These findings indicated that the proposed method is a powerful tool for monitoring the key mutations in pathogens and allows for modifications to incorporate newer upcoming variants.