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

使用 DNA 引导的 Argonaut 酶高灵敏度检测突变 cf-ctDNA

基本信息

批准号：
9510886
负责人：
Haim H Bau
金额：
$ 21.01万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-03-15 至 2020-02-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9510886
关键词：
Address Alleles BRAF gene Biological Assay Blood Blood specimen Body Fluids CRISPR/Cas technology Cancer Detection Cancer Patient Cleaved cell Closure by clamp DNA DNA amplification Detection Digestion Drug resistance Effectiveness Enzymes Future Genetic Genetic Diseases Genotype KRAS2 gene Laboratories Malignant Neoplasms Malignant neoplasm of pancreas Masks Methods Monitor Mutation Nucleic Acids Oncologist PIK3CA gene Peptide Nucleic Acids Pharmaceutical Preparations Polymerase Proteins Reagent Risk Sampling Scheme Signal Transduction Techniques Thermus thermophilus Time Treatment outcome Urine base cancer biomarkers cancer cell cancer genetics cancer type cost cost effective design exhaust experimental study fetal improved individualized medicine instrument interest liquid biopsy mutant nuclease operation rare variant therapy outcome thermophilic bacteria tool

项目摘要

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.

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的癌细胞液体活检有显着改善治疗的潜力。检测体液中与癌症相关的突变 DNA，例如血液和尿液中的过量野生型 (WT) DNA 通常需要“大海捞针”的方法。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非常低丰度的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) 聚合酶扩增钳技术尽管这些策略在某些情况下取得了成功，但它们也存在放大效应。存在许多缺点，并且通常需要多个耗时的单元操作，这会增加为了增强和克服现有浓缩工具的局限性，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) 中分离出来的 TtAgo 具有高度特异性、高效性和多功能性。 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）使 TtAgo 能够进行多重切割。工作温度 > 65oC，最大限度地减少 DNA 引导的非特异性杂交，使 TtAgo 成为理想选择与各种等温扩增方案集成在该项目结束时，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）的灵敏度这里开发的方法对于增强也很有用。检测其他罕见突变等位基因，例如与胎儿异常相关的等位基因。