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， 治疗，以配合日益增长的武器库的药物与癌症遗传学，并监测基因的变化， 几乎是真实的时间内检测癌细胞。液体活检具有显著改善治疗的潜力 成果，同时降低成本。检测体液中与癌症相关的突变DNA，如 血液和尿液，往往需要“大海捞针”的办法。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， 突变等位基因的信号。富集极低丰度异常核酸的常用策略 包括用引导切割酶如CRISPR Cas9和/或 抑制WT DNA的肽核酸（PNA）聚合酶扩增钳位技术 放大虽然这些战略在某些情况下是成功的，但它们受到 许多缺点，并且通常需要多个耗时的单元操作， 污染风险。为了增强和克服现有浓缩工具的局限性，我们 提出了一种新的富集方法，利用DNA引导的argonaute蛋白裂解酶 分离自嗜热栖热菌Thermus thermophilus（TtAgo）。TtAgo是高度特异性，高效和通用的。不像 CRISPR cas9，TtAgo不需要存在任何特定序列，如PAM基序 (that是CRISPR Cas9所必需的）以实现切割。TtAgo适合于多路复用， 在> 65 ℃下运行，最大限度地减少了DNA指导的非特异性杂交，使TtAgo成为理想的 用于与各种等温扩增方案整合。在这个项目结束时，我们将 已经开发了一种基于TtAgo的用于稀有等位基因富集的多重测定， 增强下游突变等位基因检测方案如NGS和ddPCR的灵敏度 具有和不具有多路预放大。这里开发的方法也有助于提高 检测其他罕见的突变等位基因，如与胎儿异常相关的等位基因。

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.