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

Area of Science:

• Biotechnology
• Molecular Biology
• Cancer Research

Background:

• Accurate quantification of circulating tumor DNA (ctDNA) is crucial for cancer diagnostics and treatment.
• Existing ctDNA detection assays face challenges with background noise, limiting precision.

Purpose of the Study:

• To develop a novel assay for background-free and absolute quantification of ctDNA.
• To overcome limitations of existing ctDNA detection methods.

Main Methods:

• Developed a synthetic gene circuit assay termed CATCH, utilizing a multilevel switch to regulate transcription and translation, thereby eliminating background noise.
• Integrated the gene circuit with a Cas9 nickase H840A recognizer and a molecular beacon reporter.
• Implemented CATCH in a one-pot reaction at 35°C and integrated it with a digital chip for digital CATCH.

Main Results:

• CATCH demonstrated virtually no background noise, enabling robust absolute quantification of ctDNA.
• Digital CATCH achieved precise ctDNA quantification.
• Clinical validation in 76 non-small cell lung cancer patients showed high sensitivity and specificity in detecting drug-resistant ctDNA mutations.

Conclusions:

• CATCH is a simple, robust, and sensitive assay for background-free ctDNA detection and quantification.
• The CATCH assay shows significant potential as a powerful tool for next-generation ctDNA analysis in clinical settings.
• This technology can improve tumor diagnostics and treatment monitoring through accurate ctDNA measurement.