Synthetic Gene Circuit-Based Assay with Multilevel Switch Enables Background-Free and Absolute Quantification of Circulating Tumor DNA

Circulating tumor DNA (ctDNA) detection has found widespread applications in tumor diagnostics and treatment, where the key is to obtain accurate quantification of ctDNA. However, this remains challenging due to the issue of background noise associated with existing assays. In this work, we developed a synthetic gene circuit-based assay with multilevel switch (termed CATCH) for background-free and absolute quantification of ctDNA. The multilevel switch combining a small transcription activating RNA and a toehold switch was designed to simultaneously regulate transcription and translation processes in gene circuit to eliminate background noise. Moreover, such a multilevel switch-based gene circuit was integrated with a Cas9 nickase H840A (Cas9n) recognizer and a molecular beacon reporter to form CATCH for ctDNA detection. The CATCH can be implemented in one-pot reaction at 35°C with virtually no background noise, and achieve robust absolute quantification of ctDNA when integrated with a digital chip (i.e., digital CATCH). Finally, we validated the clinical capability of CATCH by detecting drug-resistant ctDNA mutations from the plasma of 76 non- small cell lung cancer (NSCLC) patients, showing satisfying clinical sensitivity and specificity. We envision that the simple and robust CATCH would be a powerful tool for next-generation ctDNA detection.