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

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

By placing the fluorophore FAM on the 5'-end and a 7-mer DNA extension on the 3'-end of the crGFP, we learned that the first Uracil on the 5'-end of the crGFP gets trimmed by LbCas12a in the absence of an activator, which corroborated previous studies reported for FnCas12a 12 . As a result, the 5'-end modifications are eliminated and converted back to the wild-type crRNA before complexing with the activator. This finding reinforces our previous observation that the 5'extended crRNA has similar collateral cleavage activity as the wild-type crRNA. Fascinated by LbCas12a pre-crRNA processing as previously described 17 and from our observations, we investigated how extensions of the mature crRNA would influence the trans-cleavage activity compared to the corresponding extended pre-crRNA. We discovered that the modified pre-crRNA and modified mature crRNA (tru-crRNA) exhibited comparable trans-cleavage efficiency (Fig. 2g) . Furthermore, when a dsDNA or an ssDNA activator was present, the 3'-and 5'-end DNA- To further understand the LbCas12a enhanced enzymatic activity, we performed a Michaelis-Menten kinetic study on the wild-type crGFP and the crGFP+3'DNA7 and observed that the ratio Kcat/Km was 3.2-fold higher for crGFP+3'DNA7 than the unmodified crGFP (Figs. 2c,d) . The time-dependent gel electrophoresis analysis of nonspecific cleavage of ssDNA M13mp18 phage (~7 kb) reconfirmed the fluorophore-quencher-based reporter assay results (Fig. 2e) .