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The copyright holder for this preprint (which was not peer-reviewed) is the .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 However, the two reaction rates eventually reach a similar saturation point (Supplementary Figs. [16] [17] [18] . This suggests that Mg 2+ is not only required for the Cas12a reaction, but also accelerates the enzyme's trans-cleavage activity. Regardless, Mg 2+ plays an important role in lowering the limit of detection in synthetic urine containing PCA3. While as low as 25 fM (equivalent to 2.5 attomoles) of PCA3 cDNA can be detected with ENHANCE without any target amplification ( Supplementary Fig. 19) , the clinical concentration of PCA3 mRNA in the urine can be lower and therefore may require target pre-amplification. 25, 26 Therefore, we incorporated and tested a recombinase polymerase amplification (RPA) step to isothermally amplify the PCA3 cDNA. By combining the RPA step as previously reported, 1,7 the concentration of PCA3 cDNA in the urine was detectable down to ~10 aM (1 zmol) with 2.9-fold signal to noise ratio (Fig. 3d) .