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.

中文翻译：

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通过Cas9 / sgRNA消化和PCR扩增富集后，提高了EGFR突变检测的敏感性

摘要

循环肿瘤DNA的检测在癌症研究和临床实践中很重要。在本研究中，我们旨在使用簇状规则间隔的短回文重复序列（CRISPR）/ CRISPR相关蛋白9（Cas9）系统来选择性靶向野生型片段，以提高下一代测序下游突变检测的敏感性对突变片段的切割活性低或没有切割活性，然后使用聚合酶链反应进行扩增。我们选择了表皮生长因子受体基因（EGFR）的不同突变位点）-exon19缺失的肺癌患者，并构建了比例为10％至0.01％的突变和野生型DNA混合模板，以测试富集方法的有效性。结果表明，在CRISPR / Cas9富集后，通过Sanger测序可以检测到低浓度的突变DNA片段（0.01％），与未处理的样品相比，增加了1000倍。我们进一步验证了该方法的可行性，并在非小细胞肺癌患者的临床样本中获得了相似的结果，表明该方法具有在早期检测低拷贝数突变的潜力。