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

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

The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.04.13.036079 doi: bioRxiv preprint commercially available paper-based lateral flow assay with a FITC-ssDNA-Biotin reporter, [27] [28] [29] we could visually detect 1 nM of CoV-2 cDNA within 20 minutes of incubation using both wildtype and modified crRNAs without any target amplification (Fig. 3j-l and Supplementary Fig. 26 ). The enzyme trans-cleavage activity exhibited a consistent trend with the crRNA+3'DNA7 among five different targets (Fig. 3f) . When incorporating a reverse transcription step and a loop-mediated isothermal amplification (RT-LAMP) strategy into the ENHANCE, both the crCoV-2-WT and the crCoV-2+3'DNA7 demonstrated a limit of detection down to a 3-300 copies of RNA (Fig. 3m) . However, in case of crCoV-2-WT, the partial cleavage of the reporter resulted in a darker control line on the paper strip. Band-intensity analysis showed that the ENHANCE exhibited an average of 23-fold higher ratio of positive to control line between 1 nM and 1 pM of target CoV-2 RNA, while the crCoV-2-WT indicated an average of only 7-fold ratio (Figs. 3m,n and Supplementary Fig. 27) . Additionally, the time lapse pictures of lateral flow assays showed that the positive line for target-containing samples developed and became visible sooner (within 30 seconds) for the crCoV-2+3'DNA7 than the crCoV-2-WT ( Supplementary Fig. 28 ).