Integration of AND logic circuit with CRISPR/Cas12a system for sensitive detection of biomarkers and accurate discrimination of breast cancer cells.

Flap endonuclease 1 (FEN1) is a structure-selective nuclease that is of great significance in maintaining genomic stability. FEN1 is up-regulation in various cancers and is regarded as a new biomarker for cancer diagnosis. Herein, we integrate DNA logic circuit with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system for sensitive detection of FEN1 at the cellular level and accurate discrimination of different cell types. In this design, FEN1 cleaves the flap-containing synthetic substrates, and subsequently magnetic separation separates the primers from the cleaved substrate fragments, inducing the release of the primers. The released primers can initiate strand displacement amplification (SDA) reaction to generate abundant activators that can activate the trans-cleavage activity of the CRISPR/Cas12a system to cleave the signal probes for the recovery of Cy5 fluorescence signal. This circuit displays superior sensitivity with a detection limit of 5.19 × 10 U/μL, and it is capable of screening the FEN1 inhibitors, quantifying the activity of FEN1 with sensitivity at the single-cell level, and discriminating diverse FEN1 levels in clinical breast cancer tissues. We further construct an AND logic circuit based on miR-31 and FEN1 to simultaneously monitor the biomarkers at the cellular level and achieves accurate discrimination of different breast cancer cell types with a P-value of less than 0.05. The integration of DNA logic circuit with CRISPR/Cas12a system provides a new approach for biomarker-related biomedical research and clinical diagnostics.