High Sensitivity Detection of Mutant cf-ctDNA with DNA-Guided Argonaut Enzymes

液体活检正在成为一种强大的、具有成本效益的工具，用于对癌细胞进行基因分型、个体化治疗 将不断增长的药物与癌症遗传学相匹配的疗法，并监测基因变化 几乎实时地检测癌细胞。液体活检有显着改善治疗的潜力 成果，同时降低成本。体液中与癌症相关的突变DNA的检测，例如 血液和尿液，往往需要“大海捞针”的方法。过量的野生型 (WT) DNA 在聚合酶扩增过程中耗尽必需试剂，引入假阳性和掩模 突变等位基因的信号。富集极低丰度异常核酸的常见策略 包括使用 CRISPR Cas9 等引导切割酶选择性消化 WT DNA 和/或 抑制 WT DNA 的肽核酸 (PNA) 聚合酶扩增钳技术 放大。尽管这些策略在某些情况下取得了成功，但它们也面临着以下问题： 存在许多缺点，并且通常需要多个耗时的单元操作，这会增加 污染风险。为了增强和克服可用丰富工具的局限性，我们 提出了一种利用 DNA 引导的阿尔古特蛋白裂解酶的新富集方法 从嗜热栖热菌 (TtAgo) 中分离出来。 TtAgo 具有高度特异性、高效性和多功能性。不像 CRISPR cas9、TtAgo 不需要存在任何特定序列，例如 PAM 基序 （这对于 CRISPR Cas9 至关重要）以实现切割。 TtAgo 适合多路复用和 工作温度 > 65oC，最大限度地减少 DNA 引导的非特异性杂交，使 TtAgo 成为理想选择 用于与各种等温扩增方案集成。在这个项目结束时，我们将 开发了一种基于 TtAgo 的多重检测，用于稀有等位基因的富集，这将极大地促进稀有等位基因的富集。 增强下游突变等位基因检测方案（例如 NGS 和 ddPCR）的灵敏度 有或没有多路预放大。这里开发的方法也可用于增强 检测其他罕见突变等位基因，例如与胎儿异常相关的突变等位基因。

Liquid biopsy is emerging as a powerful, cost effective tool for genotyping cancer cells, individualizing therapy to match the growing arsenal of drugs with cancer genetics, and monitoring genetic shifts in cancer cells in nearly real time. Liquid biopsy has the potential of significantly improving therapy outcomes while reducing cost. The detection of cancer-related mutant DNA in body fluids, such as blood and urine, often requires a “needle in a haystack” approach. Excess wild-type (WT) DNA exhausts essential reagents during polymerase amplification, introduces false positives, and masks mutation alleles’ signals. Common strategies to enrich very low abundance aberrant nucleic acids include selective digestion of WT DNA with guided cleaving enzymes such as CRISPR Cas9 and/or peptide nucleic acid (PNA) polymerase amplification clamping technique that suppresses WT DNA amplification. Although these strategies have been successful in some cases, they suffer from a number of shortcomings and often require multiple, time-consuming unit operations that increase contamination risk. To augment and overcome the limitations of available enrichment tools, we propose a new enrichment method that utilizes the DNA-guided, argonaute protein cleaving enzyme isolated from Thermus thermophilus (TtAgo). TtAgo is highly specific, efficient, and versatile. Unlike CRISPR cas9, TtAgo does not require presence of any specific sequences such as the PAM motif (that is essential for CRISPR Cas9) to enable cleaving. TtAgo is amenable to multiplexing and operates at > 65oC, which minimizes non specific hybridization of DNA guides and makes TtAgo ideal for integration with various isothermal amplification schemes. At the conclusion of this project, we will have developed a TtAgo-based multiplexed assay for rare alleles’ enrichment that would greatly enhance the sensitivity of downstream mutant allele detection schemes such as NGS and ddPCR with and without multiplexed pre-amplification. The method developed here is also useful to enhance detection of other rare mutant alleles such as associated with fetal abnormalities.