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、经济高效的癌症细胞基因分型工具，个性化 将不断增长的药物武器库与癌症遗传学相匹配的治疗方法，并监测 近乎实时的癌细胞。液体活组织检查有可能显著改善治疗 在降低成本的同时取得成果。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。过量野生型(WT)DNA 在聚合酶扩增过程中耗尽必要的试剂，引入假阳性，并屏蔽 突变等位基因的信号。富含极低丰度异常核酸的常用策略 包括利用诸如CRISPR Cas9和/或CRISPR Cas9和/或 抑制WTDNA的PNA聚合酶扩增钳制技术 放大。尽管这些策略在某些情况下取得了成功，但它们受到了 许多缺点，而且往往需要多次、耗时的单元操作，从而增加 污染风险。为了增强和克服现有丰富工具的局限性，我们 提出了一种利用DNA引导的ArgAerte蛋白裂解酶进行富集的新方法 分离自嗜热嗜热菌(TtAgo)。TtAgo具有高度的特异性、高效性和多功能性。不像 CRISPR Cas9，TtAgo不需要任何特定序列的存在，如PAM基序 (这对于CRISPR Cas9来说是必不可少的)以启用切割。TtAgo适用于多路传输和 在&gt；65oC运行，最大限度地减少DNA指南的非特异性杂交，使TtAgo成为理想的 用于与各种等温放大方案集成。在这个项目结束时，我们将 我开发了一种基于TtAgo的稀有等位基因富集法，这将极大地 提高下游突变等位基因检测方案的灵敏度，如NGS和ddPCR 具有和不具有多路前置放大。这里开发的方法也有助于增强 检测其他罕见的突变等位基因，如与胎儿畸形有关。

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.