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Dissecting contribution of Cas9-induced DNA unwinding to specificity in gene editing

剖析 Cas9 诱导的 DNA 解旋对基因编辑特异性的贡献

基本信息

批准号：
1818107
负责人：
Peter Qin
金额：
$ 70万
依托单位：
University of Southern California
依托单位国家：
美国
项目类别：
Standard Grant
财政年份：
2018
资助国家：
美国
起止时间：
2018-08-01 至 2024-07-31
项目状态：
已结题

来源：
https://www.nsf.gov/awardsearch/showAward?AWD_ID=1818107&HistoricalAwards=false
关键词：
Dissecting contribution Cas9 induced DNA

项目摘要

The groundbreaking 2013 discovery that CRISPR-Cas9 can be programmed to efficiently edit genomes in complex organisms sparked a revolution in genetic engineering that is still rapidly unfolding. This project will test a hypothesis on how CRISPR-Cas9 discriminates between correct vs. incorrect DNA targets, and advance the capability of a biophysical technique known as site-directed spin labeling to the level of interrogating biomolecules one at a time under physiological conditions. The research is expected to yield mechanistic understanding that will aid further development of genome editing tools, and to advance technology that can benefit the broader scientific community. The project will also contribute to training of the STEM workforce via research engagement and curriculum development at both graduate and undergraduate levels, and will support outreach to the public on issues related to genome editing that have profound societal impacts.Clustered-Regularly-Interspaced-Short-Palindromic-Repeats (CRISPR) and CRISPR-associated (Cas) proteins provide adaptive immunity for bacteria and archaea. In type II CRISPR, a single Cas9 protein is activated by small RNA(s) to cleave DNA duplexes at specific sites. A key step in Cas9 target acquisition is unwinding of the DNA duplex to form a stable R-loop structure in which the RNA guide-segment is base-paired with the target-strand of the DNA protospacer. Studies have established a sequential unwinding model for Cas9 target acquisition, and these mechanistic insights have been instrumental in driving developments of CRISPR-based technology. However, understanding of mechanisms of Cas9 function is far from complete. In this project, Aim 1 will map the boundary of Cas9-induced DNA unwinding in bulk solution using a combination of spin-labeling and fluorescence spectroscopy; Aim 2 will examine the interdependency between Cas9 target cleavage and variations in the boundary of DNA unwinding; and Aim 3 will develop spin-labeling methods for investigating Cas9-induced DNA unwinding at the level of single molecules. The work will advance fundamental understanding of CRISPR-Cas9 function by testing a "two-state equilibrium" hypothesis on target discrimination, and will yield new biophysical techniques that broadly impact studies on nucleic acids and protein-nucleic acid complexes.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.

2013年的突破性发现，CRISPR-Cas9可以被编程以有效地编辑复杂生物体中的基因组，引发了一场仍在迅速展开的基因工程革命。该项目将测试CRISPR-Cas9如何区分正确与不正确的DNA靶标的假设，并将生物物理技术（称为定点自旋标记）的能力提升到在生理条件下一次一个地询问生物分子的水平。这项研究预计将产生机制上的理解，这将有助于基因组编辑工具的进一步发展，并推动技术的发展，使更广泛的科学界受益。该项目还将通过研究参与和研究生和本科生水平的课程开发，为STEM劳动力的培训做出贡献，并将支持就具有深远社会影响的基因组编辑相关问题向公众进行宣传。CRISPR和CRISPR相关蛋白为细菌和古细菌提供适应性免疫。在II型CRISPR中，单个Cas9蛋白被小RNA激活以在特定位点切割DNA双链体。Cas9靶标获取的关键步骤是DNA双链体解旋以形成稳定的R环结构，其中RNA引导片段与DNA前间隔区的靶链碱基配对。研究已经建立了Cas9靶标获取的顺序解旋模型，这些机制见解有助于推动基于CRISPR的技术的发展。然而，对Cas9功能机制的理解还远未完成。在这个项目中，Aim 1将使用自旋标记和荧光光谱的组合来绘制Cas9诱导的DNA解旋在本体溶液中的边界; Aim 2将检查Cas9靶切割与DNA解旋边界变化之间的相互依赖性; Aim 3将开发自旋标记方法，用于在单分子水平上研究Cas9诱导的DNA解旋。这项工作将通过测试关于靶点识别的“双态平衡”假设来推进对CRISPR-Cas9功能的基本理解，并将产生新的生物物理技术，广泛影响核酸和蛋白质-核酸复合物的研究。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。