Improved EGFR mutation detection sensitivity after enrichment by Cas9/sgRNA digestion and PCR amplification.

Abstract

The detection of circulating tumor DNA is important in cancer research and clinical practice. In the present study, we aimed to improve the sensitivity of downstream mutation detection of next-generation sequencing using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system to selectively target wild-type fragments but with low or no cleavage activity to mutant fragments, followed by amplification using polymerase chain reaction. We selected different mutant sites of epidermal growth factor receptor gene (EGFR)-exon19 deletions in patients with lung cancer and constructed mixed templates of mutant and wild-type DNA comprising ratios of 10% to 0.01% to test the effectiveness of the enrichment method. The results showed that after CRISPR/Cas9 enrichment, a low concentration of mutant DNA fragments (0.01%) could be detected by Sanger sequencing, which represented a 1000-fold increase compared with the untreated samples. We further verified the feasibility of the introduced method and obtained similar results in clinical samples from patients with non-small cell lung cancer, indicating that this method has the potential to detect low copy number mutations at the early stage.