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

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.

The Association of miR-let 7b and miR-548 with PTEN in Prostate Cancer.

PURPOSE: This study aims to investigate the expression level of mir-let7b-3p and mir-548, which are involved in PTEN expression in tissue samples of prostate cancer patients versus benign prostate hyperplasia (BPH) and normal adjacent tissue. MATERIALS AND METHODS: Prostate cancer tissues were obtained from patients after receiving informed consent. Total RNA extraction and cDNA synthesis were performed for determining gene expression. RESULTS: Ten patients were determined to have high Gleason scores (> 7), 36 and seven samples had intermediate Gleason scores (7?) and BPH, respectively, and 40 samples were derived from normal adjacent tissue. Downreg-ulation of mir-let7b and upregulation of mir-548 expression significantly correlated with high-risk Gleason scores. CONCLUSION: The present study showed that miR-let7b and/or mir-548 can be considered as potential targets in prostate cancer therapy.