CRISPincette, and detection of low frequency cancer mutations in ctDNA.

Abstract

61 Background: Liquid biopsy has emerged as a non-invasive and widely applicable approach in clinical practice for cancer detection and monitoring. Detecting low-frequency mutations in circulating tumor DNA (ctDNA) is crucial for the success of liquid biopsy; however, technical limitations have hindered the detection of low variant allele frequency (VAF) mutations. To address this issue, we developed CRISPincette, a novel method which combines next-generation sequencing with an advanced CRISPR system, GeneCker-Cas9. Methods: CRISPincette was designed to efficiently detect mutant alleles by eliminating abundant wild-type alleles from normal cell-induced cell-free DNA (cfDNA) using CRISPR/Cas9. An engineered CRISPR/Cas9 variant, GeneCker-Cas9, was developed to maintain single-base precision outside the PAM region, enabling efficient discrimination of single-base mutations at all 20 positions within a single guide RNA target sequence. This advanced fidelity significantly improved the accuracy compared to high-specificity SpCas9 variants (SpCas9-HF and eSpCas9). To validate the sensitivity and accuracy of ctDNA detection using CRISPincette, we employed NGS standard materials (Horizon HD776, 778, 779) and assessed the improvement in ctDNA detection sensitivity for EGFR gene mutations (EGFR 19del, EGFR L858R, EGFR T790M). Subsequently, we analyzed the clinical applicability of CRISPincette using plasma samples from 7 non-small cell lung cancer (NSCLC) patients (6 patients with EGFR mutations and 1 patient without EGFR mutations). Results: Our analysis showed an approximately 10 to 30-fold enhancement in the detection sensitivity of EGFR gene mutations. Concordant detection of EGFR mutations was observed in the 6 patients with EGFR mutations using real-time PCR tests in each tissue biopsy, and no EGFR mutation was detected in the patient without the EGFR mutation. This confirmed CRISPincette's high accuracy and sensitivity in detecting low-frequency mutations in ctDNA. Conclusions: The implementation of CRISPincette has significantly improved the accuracy and sensitivity of liquid biopsy, making it a reliable and non-invasive alternative for confirming molecular pathological information in cancer patients. Furthermore, the high accuracy of CRISPincette will be a powerful tool for monitoring treatment and prognosis through the timing of anticancer drug administration. Overall, CRISPincette represents a substantial advancement in liquid biopsy technology, with potential implications for cancer diagnosis, treatment allocation, and monitoring the emergence of resistance.