CAREER: CRISPR-based biosensors for the ultra-accurate detection of disease-related single nucleotide polymorphisms (SNPs)

职业：基于 CRISPR 的生物传感器，用于超准确检测与疾病相关的单核苷酸多态性 (SNP)

• 批准号: 2339868
• 负责人: Juhong Chen
• 金额: $ 54.88万
• 依托单位: Virginia Polytechnic Institute and State University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2024
• 资助国家: 美国
• 起止时间: 2024-07-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2339868&HistoricalAwards=false
• 关键词: CAREER; CRISPR; based; biosensors; ultra

Single nucleotide polymorphisms (SNPs) are the most common types of genetic variations in the human genome. Each SNP represents a difference in a single DNA building block, called a nucleotide. With the completion of the human genome project, more and more SNPs have been identified as biomarkers for life-threatening diseases. Detecting such SNPs is of particular importance to the fields of cancer diagnosis, pathogen detection, and various health condition monitoring. The standard methods to detect SNPs rely on real-time polymerase chain reaction (PCR) and next-generation sequencing (NGS), but are largely limited by their poor sensitivity and bad accuracy. This CAREER project will exploit the newly discovered CRISPR technology to detect disease-related SNPs, improving human health. To provide broader impacts on a national level, this project will pursue the following two education and outreach goals: 1) broaden participation in careers in biotechnology and 2) equip undergraduate and graduate students with the required biotechnology knowledge and hands-on experience. In bacteria and archaea cells, CRISPR was developed as an adaptive defense system against viral infections. In addition to gene editing, CRISPR technology has been applied to detect nucleic acids in the past few years. This NSF CAREER project aims to study the fundamental properties of CRISPR Cas nucleases through their non-specific collateral activities (also named “trans-cleavage” activity) and apply the knowledge to detect disease-related SNPs. The central hypothesis is that systematic characterization and optimization of the trans-cleavage activities of the CRISPR Cas nucleases (Cas12, Cas13, and Cas14) will provide a solid foundation to develop novel biosensing approaches for the ultra-accurate detection of disease-related SNPs (less than 0.1% SNPs in wild type can be detected). The major research objectives are: 1) enable an ultra-accurate detection of SNPs using a CRISPR-Cas14 biosensor; 2) design a ratiometric detection of SNPs using biorthogonal CRISPR-Cas13 systems; and 3) demonstrate CRISPR-12 array sensors to identify multiple SNPs simultaneously.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

单核苷酸多态性（SNP）是人类基因组中最常见的遗传变异类型。每个SNP代表一个称为核苷酸的单个DNA构建块的差异。随着人类基因组项目的完成，越来越多的SNP被确定为威胁生命疾病的生物标志物。检测这种SNP对于癌症诊断，病原体检测和各种健康状况监测的领域尤其重要。检测SNP的标准方法依赖于实时聚合酶链反应（PCR）和下一代测序（NGS），但在很大程度上受其敏感性和准确性不佳的限制。该职业项目将利用新发现的CRISPR技术来检测与疾病相关的SNP，从而改善人类健康。为了对国家一级产生更广泛的影响，该项目将追求以下两个教育和推广目标：1）扩大对生物技术职业的参与，以及2）为本科生和研究生提供所需的生物技术知识和实践经验。在细菌和古细胞细胞中，CRISPR是一种针对病毒感染的自适应防御系统。除基因编辑外，在过去几年中，还采用了CRISPR技术来检测核酸。这个NSF职业项目旨在通过其非特异性附带活动（也称为“反式裂解”活动）来研究CRISPR CAS核的基本特性，并应用知识来检测与疾病相关的SNP。中心假设是，CRISPR CAS核者的反裂解活性的系统表征和优化（CAS12，CAS13和CAS14）将为开发新型的生物传感方法提供稳固的基础，以检测到与疾病相关的SNP的超清晰度检测（在野外类型中少于0.1％的SNP）。主要的研究目标是：1）使用CRISPR-CAS14生物传感器对SNP进行超准确的检测； 2）使用生物息肉CRISPR-CAS13系统设计了SNP的比率检测； 3）展示CRISPR-12阵列传感器简单地识别多个SNP。该奖项反映了NSF的法定任务，并被认为是值得通过基金会的知识分子优点和更广泛的影响审查标准的评估来支持的。