A "turn-on" CRISPR-mediated method using enhanced fluorescent bimetallic DNA nanoclusters for EGFR mutation detection
Fatemeh Goudarzi1, Pouya Salehipour2, Mohammad Hossein Modarressi3
1Nanobiosensors Lab, Department of Nanobiotechnology and Biomimetics, School of Life Science Engineering, College of Interdisciplinary Science and Technology, University of Tehran, Tehran, Iran.
Abstract:
An affordable, precise detection of mutations is critical for guiding targeted cancer therapies and improving patient outcomes. Epidermal growth factor receptor (EGFR), a protein on the surface of cells that regulates growth and division, is frequently mutated in non-small cell lung cancer (NSCLC). Early identification of these mutations enables clinicians to select the most effective tyrosine kinase inhibitors, thereby enhancing treatment response and survival rates. Recent studies have focused on developing CRISPR-based detection strategies incorporating nanomaterials to achieve more accurate results. In this study, we present a CRISPR-based "turn-on" detection platform that leverages the cleavage of a novel enhanced bimetallic DNA nanocluster to measure EGFR exon 19 deletion in non-small cell lung cancer (NSCLC). The system is innovatively designed using guide RNAs (gRNAs) rationally derived from the normal EGFR gene, enabling the determination of exon 19 deletion through CRISPR-Cas activation in samples containing the normal and mutant. Upon recognition of the normal EGFR gene, the Cas12a enzyme induces cleavage of the Spermiform-designed Ag/Au DNA nanocluster and fluorescence quenching. At the same time, fluorescence signal retention depends on mutation frequency, with higher mutation frequencies resulting in greater or "turn-on" fluorescence signals. This approach achieves a detection limit (LOD) of approximately 0.35 nM, which is capable of detecting about 1.5% mutation, offering a cost-effective, label-free diagnostic tool and a promising strategy for future detection of deletion-related subtypes in PCR products by targeting normal sequences. The integration of bimetallic nanocluster-based reporters with CRISPR precision provides an emerging platform for next-generation molecular diagnostics targeting EGFR and other clinically relevant mutations.
Insights
A new CRISPR-based platform precisely detects epidermal growth factor receptor (EGFR) exon 19 deletions in non-small cell lung cancer (NSCLC). This affordable, label-free method uses bimetallic DNA nanoclusters for enhanced diagnostic accuracy.
Area of Science:
- Molecular Diagnostics
- Biotechnology
- Genomics
Background:
- Epidermal growth factor receptor (EGFR) mutations are key drivers in non-small cell lung cancer (NSCLC), necessitating precise detection for targeted therapy.
- Current diagnostic methods for EGFR mutations can be costly and lack the sensitivity required for early detection.
- CRISPR-Cas systems combined with nanomaterials offer a promising avenue for developing sensitive and specific molecular diagnostic tools.
Purpose of the Study:
- To develop and validate a novel CRISPR-based "turn-on" detection platform for the precise identification of EGFR exon 19 deletions in NSCLC.
- To leverage enhanced bimetallic DNA nanoclusters and CRISPR-Cas12a for a sensitive and cost-effective diagnostic assay.
- To establish a label-free detection system capable of quantifying mutation frequencies.
Main Methods:
- A CRISPR-Cas12a system was designed using guide RNAs targeting the normal EGFR gene.
- A novel enhanced bimetallic Ag/Au DNA nanocluster was synthesized as a reporter molecule.
- The system detects EGFR exon 19 deletion by monitoring fluorescence changes upon CRISPR-Cas12a activation and nanocluster cleavage.
Main Results:
- The platform achieved a low limit of detection (LOD) of approximately 0.35 nM, enabling the detection of ~1.5% mutation.
- The system demonstrated a "turn-on" fluorescence signal proportional to the mutation frequency.
- The assay is label-free and cost-effective, offering a significant advantage over existing methods.
Conclusions:
- The developed CRISPR-based platform provides an accurate and affordable method for detecting EGFR exon 19 deletions in NSCLC.
- This approach, integrating CRISPR precision with bimetallic nanoclusters, represents a promising next-generation molecular diagnostic tool.
- The technology holds potential for broader applications in detecting other clinically relevant mutations and deletion-related subtypes.
