Split activator ligation assay: A CRISPR/Cas12a-enabled biosensor for the genotyping of cancer-associated miRNA single-nucleotide polymorphisms

Abstract

Although assays for microRNA (miRNA) detection have been largely developed and applied, the identification of single-nucleotide polymorphisms in miRNAs (miR-SNPs), which are emerging cancer biomarkers, remains a significant challenge. The rs11614913 SNP in miR-196a2, resulting in the mutant miR-196a2T variant with a single nucleotide change from the wildtype miR-196a2C, has been linked to cancer diagnosis. In this work, we present the split activator ligation assay (SALA) for miR-SNP genotyping, marking the first use of CRISPR/Cas12a in this context. In SALA, miR-196a2T facilitates the ligation of split Cas12a activators via SplintR ligase, leading to the full activation of Cas12a and subsequent fluorescence generation. SALA detects miR-SNPs at femtomolar concentrations, allowing for the precise detection of SNPs when the wildtype allele is in over 2000-fold excess. Analyses of clinical samples demonstrate the robustness of SALA in distinguishing cancerous from non-cancerous samples, underscoring its potential utility in cancer diagnostics.