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 BLI Ni-NTA biosensors were purchased from Fortebio to perform the binding kinetic study with polyhistidine-tagged LbCas12a. In detail, the experiment was carried out in a 96-well plate and included five steps: baseline, loading, baseline2, association, and dissociation. The biosensors were dipped into the baseline containing 1X kinetic buffer (1X PBS, 0.1% BSA, and 0.01% Tween 20). They were then transferred into each loading well containing 10 g/ml of LbCas12a. After processing through loading and baseline2, the protein-tagged biosensor was next allowed to dip into the crRNA sample wells at different dilution (10, 5, 2.5, 1.25, 0.625, 0.3125, 0.15625, and 0 g/ml) in the association step. The dissociation step occurred when the biosensors were transferred back to baseline2 at a shake speed of 1000 rpm. All the samples were read by the Octet QKe system (Fortebio). Kd was determined by software Data Analysis 10.0 (Fortebio), and only Kd with R 2 >0.9 were extracted for comparison between crRNA wild type and modified crRNAs.