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 代表单个 DNA 构建块（称为核苷酸）的差异。随着人类基因组计划的完成，越来越多的 SNP 被识别出来。检测此类 SNP 对癌症诊断、病原体检测和各种健康状况监测领域特别重要。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），但很大程度上受到其灵敏度差和准确性差的限制，该 CAREER 项目将利用新发现的 CRISPR 技术来检测与疾病相关的 SNP，从而在国家层面上提供更广泛的影响。该项目将追求以下两个教育和推广目标：1）扩大生物技术职业的参与度；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。在细菌和古细菌细胞中，CRISPR 被开发为适应性防御除了基因编辑之外，过去几年，CRISPR 技术还被应用于检测核酸。 NSF CAREER 项目旨在通过其非特异性附带活性（也称为 CRISPR Cas 核酸酶）研究其基本特性。 “反式切割”活性）并应用这些知识来检测与疾病相关的 SNP。中心假设是 CRISPR Cas 核酸酶（Cas12、 Cas13 和 Cas14）将为开发新型生物传感方法提供坚实的基础，以超准确地检测与疾病相关的 SNP（可以检测到野生型中低于 0.1% 的 SNP）。主要研究目标是：1）实现超精确检测。使用 CRISPR-Cas14 生物传感器超准确检测 SNP；2) 使用双正交 CRISPR-Cas13 系统设计 SNP 比率检测； 3) 展示 CRISPR-12 阵列传感器可同时识别多个 SNP。该奖项反映了 NSF 的法定使命，并通过使用基金会的智力价值和更广泛的影响审查标准进行评估，被认为值得支持。