Enhancement of trans-cleavage activity of Cas12a with engineered crRNA enables amplified nucleic acid detection

Long T. Nguyen; Brianna M Smith; Piyush K. Jain

Although there are no reports on RNA-guided RNA targeting by LbCas12a, we envisioned that an RNA can potentially be detected as a DNA/RNA heteroduplex. To test this hypothesis, we incorporated a reverse transcription step to convert RNA into cDNA/RNA heteroduplex before detecting the RNA with a trans-cleavage assay. We discovered that the RNA can only be detected if the target strand for crRNA is a DNA but not an RNA in a heteroduplex. Notably, the efficiency of the trans-cleavage activity for the DNA/RNA heteroduplex was found to be significantly lower than the corresponding ssDNA or dsDNA (Fig. 3e, Supplementary Fig. 20b) . However, the DNA/RNA heteroduplex achieved an improved enzymatic collateral activity when using the crRNA+3'DNA7 compared to the wild-type crRNA. We applied the ENHANCE to successfully detect low picomolar concentrations of HIV RNA target encoding Tat gene with our DNA/RNA heteroduplex detection strategy (Fig. 3f) . In parallel, ssDNA and dsDNA targets from HIV were also detected with much higher sensitivity compared to the wild-type crRNA within 15 to 30 minutes (Figs. 3f,g and Supplementary Figs. 21) . We further applied the ENHANCE for detecting HCV ssDNA and HCV dsDNA gene encoding a polyprotein precursor, both of which indicated consistent enhanced collateral activity than the wild-type crRNAs within 24 minutes (Figs. 3f, 3h , and Supplementary Fig. 22 ). The limit of detection for HIV and HCV targets were calculated to be 700 fM cDNA and 290 fM ssDNA, respectively.