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. [Display omitted] • Enhanced trans -cleavage activity of Cas12a and Cas13a by chemical enhancers. • Sulfhydryl reductants and non-ionic surfactants are two types of chemical enhancers. • The mechanism of this enhancement is the conformation change of Cas RNP. • The reaction time of chemical enhanced CRISPR/Cas system reduced 75–83%. • The sensitivity of chemical enhanced CRISPR/Cas system increased 4–6 folds. [ABSTRACT FROM AUTHOR]