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 fluorophore-quencher reporter assay was carried out following a standard clinical detection protocol. The Cas12a-crRNA ribonucleoprotein complex was assembled by mixing 100 nM Cas12a and 100 nM crRNA in 1X NEBuffer 2.1 in the Proflex PCR system (ThermoFisher Scientific) at 25 o C for 15 minutes (volume 28.5 µL). The activator (1 nM final concentration), FQ reporter (500 nM final concentration), and UltraPure™ DNase/RNase-Free distilled water (Invitrogen) were pre-added to a 96-well plate (Greiner Bio-One) to a volume of 71.5 µL. The reaction was initiated by adding the Cas12a-crRNA mixture to the 96-well plate preloaded with activator and FQ reporter (Integrated DNA Technologies Inc). The plate was quickly transferred to a plate reader (ClarioStar or BioTek), and fluorescence intensity was measured every 3 minutes for 3 or 12 hours (detection limit assay) (FAM FQ: λex: 483/30 nm, λem: 530/30 nm; HEX: λex: 430/20 nm, λem: 555/30 nm). After 3 or 12 hours (detection limit assay), the sample was scanned for images using the Amersham Typhoon (GE Healthcare).