Increasing trans-cleavage catalytic efficiency of Cas12a and Cas13a with chemical enhancers: Application to amplified nucleic acid detection

The exceptional programable trans-cleavage ability of type V and VI CRISPR/Cas nucleases paved the way for ultrasensitive CRISPR/Cas based sensing of nucleic acid and alternative targets.However, the enhancement of the trans-cleavage activity of Cas effector with organic chemical agents has not been explored thus far.We report here chemically enhanced trans-cleavage activity of Cas12a and Cas13a nucleases which improves sensor performance in CRISPR/Cas biosensing.Improved trans-ssDNA cleavage of Cas12a and trans-ssRNA cleavage of Cas13a were demonstrated by using sulfhydryl reductants and non-ionic surfactants.DTT and PVA were demonstrated to be the most effective chemical enhancers in both cases.By using a fluorescence resonance energy transfer (FRET)-based intramolecular distance measurements, we identified the mechanism of this enhancement to be the conformation change of the ribonucleoprotein and quantified it to be major (about 50% increase of a relevant intramolecular distance).These chemical enhancers have been integrated into the established CRISPR/Cas biosensing protocols without additional modifications.For the detection of Helicobacter Pylori DNA and SARS-CoV-2 RNA, we found a decreased reaction time by 75-83% and 4-6-fold increased sensitivity.These results indicate that chemical enhancers provide a versatile and broadly applicable approach to break through the barriers of long reaction time and sensitivity in CRISPR/Cas sensors.