An exonuclease protection and CRISPR/Cas12a integrated biosensor for the turn-on detection of transcription factors in cancer cells.

Transcription factors (TFs) are critical proteins that regulate the expression of genes, and the abnormal change of TFs levels is directly related to physical dysfunctions. Herein, we developed a clustered regularly interspaced short palindromic repeats (CRISPR)-based biosensor for the measurement of TFs level with the assistance of exonuclease protection assay. A dsDNA (activator) with the ability to activate Cas12a was engineered to contain TFs binding domain, and the binding between TFs and the activator can protect the dsDNA from being digested by exonuclease III (Exo III). The reserved activator then triggered a CRISPR/Cas12a reporting reaction to produce fluorescent signal for detection. In the detection of nuclear factor-kappa B (NF-κB) p50 subunit, the limit of detection of 0.2 pM and limit of quantification of 0.6 pM were obtained respectively, and the performance of this biosensor has been challenged by cell nucleoprotein extracts. Additionally, this method can be applied in the screening and evaluation of TFs inhibitors, calculating the IC50 of oridonin. Integrating merits including high sensitivity, low cost, and good portability, this method may enrich the arsenal for TFs-related applications.