CAREER: High bandwidth nano-transistors to understand the kinetic basis for CRISPR/CAS enzymes to enhance their applications for diagnostics and therapeutics

职业：高带宽纳米晶体管，以了解 CRISPR/CAS 酶的动力学基础，以增强其在诊断和治疗方面的应用

• 批准号: 2427540
• 负责人: Kiana Aran
• 金额: $ 54.95万
• 依托单位: University of California-San Diego
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-11-01 至 2026-01-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2427540&HistoricalAwards=false
• 关键词: CAREER; bandwidth; nano; transistors; understand

The knowledge of how biological molecules interact with each other is essential to understanding their roles in sustaining life. Molecular interactions are commonly measured using optical techniques and specialized optical labels on the molecules of interest. Recent advances in modern electronics and nanoscale materials potentially allow continuous monitoring of molecular interactions in a more natural state without optical labels and instruments. The goal of this CAREER project is to develop a single-molecule high-speed nanoelectronic platform to better understand the function of CRISPR (clustered regularly interspaced short palindromic repeats)-associated enzymes known as "molecular scissors". These enzymes allow gene editing and have revolutionized many basic and applied research areas. The knowledge gained will be beneficial for many applications in CRISPR engineering, pharmaceutical drug discovery, clinical diagnostics, and agricultural science. The project will promote early research involvement and mentorship opportunities to a new generation of engineers and scientists pursuing a career of interdisciplinary research intersecting modern biology, nanotechnology and engineering.The investigator’s scientific career vision is to explore the utility of nano-electronic systems to develop transformative and customizable biosensing platforms for pharmaceutical, clinical, and environmental applications. As part of this vision, this project focuses on the integration of CRISPR (clustered regularly interspaced short palindromic repeats) with high bandwidth graphene field effect transistors (gFETs) designed for single-molecule sensing. The platform provides unique electronic signatures representing the molecular interactions that happen concurrently between the CRISPR and target DNA/RNA at different timescales. The CRISPR-Cas system is a family of RNA guided enzymes that is widely used for gene editing as it is capable of double-stranded DNA binding and cleavage, producing insertions and deletions (INDELs) at specific loci within the genome in vivo. The application areas of CRISPR technology are rapidly extending beyond genome editing, such as targeted gene regulation, in vivo imaging, epigenetic modulation as well as nucleic acid detection for diagnostic applications. The goal of this CAREER proposal is to provide a tool to evaluate the enzymatic activity of newly discovered or engineered CRISPR-Cas enzymes to better understand their biology and the impact of CRISPR-Cas mutagenesis, guide RNA modifications as well as genetic variation of the target on their functions. Successful development of the single molecule high bandwidth gFET for CRISPR analysis coupled with trained machine learning models, will provide a tool for detailed analysis of the CRISPR interactions with its target sequence in terms of binding, cleavage, and release of the target in real-time as well as identification of the variables most important for predicting CRISPR function. The information obtained can direct the design and optimization of CRISPR-Cas enzymes toward enhancing their efficiency and safety in wide range of applications. The applications of the single-molecule high bandwidth gFET platform can be expanded for understanding the molecular interactions of other enzymes beyond CRISPR.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.

生物分子如何相互作用的知识对于理解它们在维持生命中的作用至关重要。分子相互作用通常使用光学技术和感兴趣的分子上的专用光学标记来测量。现代电子学和纳米材料的最新进展可能允许在更自然的状态下连续监测分子相互作用，而无需光学标签和仪器。这个CAREER项目的目标是开发一个单分子高速纳米电子平台，以更好地了解CRISPR（成簇的规则间隔短回文重复序列）相关酶（称为“分子剪刀”）的功能。 这些酶允许基因编辑，并彻底改变了许多基础和应用研究领域。所获得的知识将有利于CRISPR工程，药物发现，临床诊断和农业科学的许多应用。该项目将促进早期的研究参与和指导机会，以新一代的工程师和科学家追求的跨学科研究交叉现代生物学，纳米技术和工程的职业生涯。研究者的科学职业生涯的愿景是探索纳米电子系统的实用性，以开发变革和可定制的生物传感平台，用于制药，临床和环境应用。作为这一愿景的一部分，该项目的重点是将CRISPR（成簇的规则间隔短回文重复序列）与专为单分子传感设计的高带宽石墨烯场效应晶体管（gFET）相结合。该平台提供了独特的电子签名，代表了CRISPR和靶DNA/RNA之间在不同时间尺度上同时发生的分子相互作用。CRISPR-Cas系统是广泛用于基因编辑的RNA引导酶家族，因为它能够在体内基因组内的特定基因座处结合和切割双链DNA，产生插入和缺失（INDEL）。CRISPR技术的应用领域正在迅速扩展到基因组编辑之外，如靶向基因调控、体内成像、表观遗传调节以及用于诊断应用的核酸检测。该CAREER提案的目标是提供一种工具来评估新发现或工程改造的CRISPR-Cas酶的酶活性，以更好地了解它们的生物学以及CRISPR-Cas诱变、指导RNA修饰以及靶标的遗传变异对其功能的影响。用于CRISPR分析的单分子高带宽gFET的成功开发加上经过训练的机器学习模型，将提供一种工具，用于详细分析CRISPR与其靶序列的相互作用，包括结合，切割和实时释放靶标，以及识别对预测CRISPR功能最重要的变量。所获得的信息可以指导CRISPR-Cas酶的设计和优化，以提高其在广泛应用中的效率和安全性。单分子高带宽gFET平台的应用可以扩展到理解CRISPR以外的其他酶的分子相互作用。该奖项反映了NSF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。