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

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

Previous studies demonstrated that FnCas12a is a metal-dependent endonuclease, and magnesium ions are required for FnCas12a-mediated self-processing of precursor crRNA. 20 Based on these findings, we hypothesized that different metal ions may significantly affect the trans-cleavage activity of LbCas12a. This led us to test a range of divalent metal cations and discovered that most ions including Ca 2+ , Co 2+ , Zn 2+ , Cu 2+ , and Mn 2+ significantly inhibited the LbCas12a activity ( Supplementary Fig. 14) . By further investigating the Zn 2+ mediated inhibition of LbCas12a, we found that the inhibition was dose-dependent ( Supplementary Fig. 15 ). Interestingly, Ni 2+ ions showed an unusual cis-cleavage activity possibly due to its interactions with the His tags on LbCas12a ( Supplementary Fig. 14) . Among the tested divalent metal ions, the Mg 2+ ions showed the highest in vitro cis-cleavage activity, which was consistent with the literature. 20 Therefore, we characterized the effect of Mg 2+ ions on trans-cleavage activity of LbCas12a. With increasing the concentration of Mg 2+ ions, a significant increase in fluorescence signal was observed in an in vitro trans-cleavage assay. By varying the amount of Mg 2+ in the Cas12a reaction, we identified that the optimal condition of Mg 2+ was around 13 mM ( Fig. 3a-b and Supplementary Figs. [16] [17] [18] [19] .