A Sensitive and Nonoptical CRISPR Detection Mechanism by Sizing Double-Stranded λ DNA Reporter

Abstract

CRISPR-based biosensors often rely on colorimetric, fluorescent, or electrochemical signaling mechanism, which involves expensive reporters and/or sophisticated equipment. Here, we demonstrated a simple, inexpensive, nonoptical, and sensitive CRISPR-Cas12a-based sensing platform to detect ssDNA targets by sizing double-stranded λ DNA as novel report molecules. In this platform, the size reduction of λ DNA was quantified by gel electrophoresis analysis. We hypothesize that the massive trans-nuclease activity of Cas12a toward λ DNA is due to the presence of single-stranded looped structures along the λ DNA sequence. In addition, we observed a strong binding affinity between Cas12a and λ DNA, which further promotes the trans-cleavage activity and helps achieve sub-picomolar detection sensitivity, ≈100 times more sensitive than the fluorescent counterpart. The concept of utilizing the physical size change of λ DNA unlocks the possibility of using a variety of dsDNA as CRISPR reporters.