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CRISPR-Based Fluorescent Reporter (CBFR) Assay for Sensitive, Specific, Inexpensive, and Visual Detection of a Specific EGFR Exon 19 Deletion in NSCLC

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Abstract

Epidermal growth factor receptor (EGFR) is a transmembrane glycoprotein receptor with intracellular tyrosine kinase activity. Mutations in the EGFR gene, including deletions in exon 19 and the mutation L858R, induce responsiveness of non-small cell lung cancer (NSCLC) to a group of drugs known as tyrosine kinase inhibitors. Here, we report the development of the CRISPR-based fluorescent reporter (CBFR) assay including a two-step strategy combining PCR amplification and Cas12a-driven cleavage to detect the delE746_A750 subtype of EGFR exon 19 deletions. Sensitivity and specificity of the CBFR assay were analyzed with different concentrations of fluorescence reporter and different amounts of PCR product. The results demonstrated that increasing the fluorescent reporter to 4 μM and the PCR product to 5 μl enhanced sensitivity. The CBFR assay could detect EGFR exon 19 deletion even with a frequency of 1% in samples. In clinical NSCLC samples, optimized CBFR assay enabled visual detection of the delE746_A750 subtype in less than 1 h. The CBFR assay provides a sensitive, specific, and simple strategy designed based on a straightforward and inexpensive process. We suggest that the CBFR assay could serve as a diagnostic approach to detect mutations, deletions, and pathogens in underequipped laboratories and promote personalized therapeutic approaches.

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Data Availability

All data generated during this study are included in this article and supplementary information file.

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Acknowledgements

The authors express great appreciation to Medical Genetics Department at Tehran University of Medical Sciences for their assistance as well as the staff of Cancer Research Center of Imam Khomeini Hospital for the provision of non-small cell lung cancer (NSCLC) samples.

Funding

This study was supported by the Tehran University of Medical Sciences 98-3-101-45502.

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Authors and Affiliations

  1. Department of Medical Genetics, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran

    Pouya Salehipour, Mojdeh Mahdiannasser, Ghazal Sedaghat Shayegan, Mina Tabrizi & Mohammad Hossein Modarressi

  2. Department of Genetics, Science and Research Branch, Islamic Azad University, Tehran, Iran

    Kimia Shankaie

  3. Department of Medical Genetics, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran

    Majid Mojarrad

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  1. Pouya Salehipour
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Contributions

PS and MHM designed the study. GSS and KS collected the samples, and reviewed cases. PS, GSS, and KS performed the experiments. PS and MM analyzed the data statistically. PS, MM, and MHM interpreted the results. PS and MM wrote the manuscript. MT and MHM revised the manuscript. All authors have read and approved the final manuscript.

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Correspondence to Mohammad Hossein Modarressi.

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Conflict of interest

The authors have no relevant financial or non-financial interests to disclose.

Ethical Approval

This study was approved by the Ethics Committee of the Tehran University of Medical Sciences (No. IR.TUMS.MEDICINE.REC.1398.421). The procedures used in this study adhere to the tenets of the Declaration of Helsinki.

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Informed consent was obtained from all individual participants included in the study.

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Participants signed informed consent regarding publishing their data.

Research Resource Identifiers (RRID)

Software: ChromasPro RRID:SCR_000229. Database: Primer-BLAST RRID:SCR_003095. Web Application: CHOPCHOP RRID:SCR_015723.

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Salehipour, P., Mahdiannasser, M., Sedaghat Shayegan, G. et al. CRISPR-Based Fluorescent Reporter (CBFR) Assay for Sensitive, Specific, Inexpensive, and Visual Detection of a Specific EGFR Exon 19 Deletion in NSCLC. Mol Biotechnol 65, 807–815 (2023). https://doi.org/10.1007/s12033-022-00576-z

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