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

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代表了一个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被鉴定为威胁生命的疾病的生物标志物。检测这些snp对于癌症诊断、病原体检测和各种健康状况监测领域具有特别重要的意义。检测snp的标准方法依赖于实时聚合酶链反应（real-time polymerase chain reaction， PCR）和下一代测序（generation -generation sequencing， NGS），但在很大程度上受到灵敏度差和准确性差的限制。这个CAREER项目将利用新发现的CRISPR技术来检测与疾病相关的snp，改善人类健康。为了在全国范围内产生更广泛的影响，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；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是作为一种抵御病毒感染的适应性防御系统而发展起来的。除了基因编辑，CRISPR技术在过去几年也被应用于核酸检测。NSF CAREER项目旨在通过CRISPR Cas核酸酶的非特异性附带活性（也称为“trans-cleavage”活性）研究其基本特性，并将其应用于疾病相关snp的检测。研究的核心假设是，系统地表征和优化CRISPR Cas核酸酶（Cas12、Cas13和Cas14）的反式切割活性，将为开发新的生物传感方法提供坚实的基础，以超精确地检测疾病相关的snp（野生型中可以检测到不到0.1%的snp）。主要研究目标是：1)使用CRISPR-Cas14生物传感器实现对snp的超精确检测；2)利用双正交CRISPR-Cas13系统设计比例检测snp；3)演示同时识别多个snp的CRISPR-12阵列传感器。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

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.