Department of Bioengineering, Stanford University School of Medicine, Stanford, CA.
Molecular Imaging Program at Stanford, Stanford University School of Medicine, Stanford, CA.
Clin Chem. 2018 Feb;64(2):307-316. doi: 10.1373/clinchem.2017.278911. Epub 2017 Oct 16.
Cell-free DNA (cfDNA) diagnostics are emerging as a new paradigm of disease monitoring and therapy management. The clinical utility of these diagnostics is relatively limited by a low signal-to-noise ratio, such as with low allele frequency (AF) mutations in cancer. While enriching for rare alleles to increase their AF before sample analysis is one strategy that can greatly improve detection capability, current methods are limited in their generalizability, ease of use, and applicability to point mutations.
Leveraging the robust single-base-pair specificity and generalizability of the CRISPR associated protein 9 (Cas9) system, we developed a deactivated Cas9 (dCas9)-based method of minor-allele enrichment capable of efficient single-target and multiplexed enrichment. The dCas9 protein was complexed with single guide RNAs targeted to mutations of interest and incubated with cfDNA samples containing mutant strands at low abundance. Mutation-bound dCas9 complexes were isolated, dissociated, and the captured DNA purified for downstream use.
Targeting the 3 most common epidermal growth factor receptor mutations (exon 19 deletion, T790M, L858R) found in non-small cell lung cancer (NSCLC), we achieved >20-fold increases in AF and detected mutations by use of qPCR at an AF of 0.1%. In a cohort of 18 NSCLC patient-derived cfDNA samples, our method enabled detection of 8 out of 13 mutations that were otherwise undetected by qPCR.
The dCas9 method provides an important application of the CRISPR/Cas9 system outside the realm of genome editing and can provide a step forward for the detection capability of cfDNA diagnostics.
无细胞 DNA（cfDNA）诊断作为疾病监测和治疗管理的新范式正在出现。这些诊断的临床实用性受到相对较低的信噪比的限制，例如癌症中低频等位基因突变（AF）。虽然在进行样本分析之前富集稀有等位基因以增加其 AF 是一种可以大大提高检测能力的策略，但目前的方法在通用性、易用性和适用于点突变方面存在局限性。
利用 CRISPR 相关蛋白 9（Cas9）系统强大的单碱基对特异性和通用性，我们开发了一种基于失活 Cas9（dCas9）的小等位基因富集方法，能够高效地进行单靶标和多重富集。将 dCas9 蛋白与靶向感兴趣突变的单指导 RNA 复合物，并与含有低丰度突变链的 cfDNA 样本孵育。突变结合的 dCas9 复合物被分离、解离，捕获的 DNA 用于下游应用进行纯化。
针对非小细胞肺癌（NSCLC）中发现的 3 种最常见的表皮生长因子受体突变（外显子 19 缺失、T790M、L858R），我们实现了 >20 倍的 AF 增加，并通过 qPCR 在 0.1%的 AF 下检测到突变。在 18 例 NSCLC 患者衍生的 cfDNA 样本队列中，我们的方法能够检测到 13 个突变中的 8 个，而 qPCR 则无法检测到这些突变。
dCas9 方法为 CRISPR/Cas9 系统在基因组编辑领域之外提供了一个重要的应用，并且可以为 cfDNA 诊断的检测能力提供一个进步。
