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

2013年的突破性发现是，CRISPR-Cas9可以被编程为有效地编辑复杂生物体的基因组，这引发了一场基因工程革命，这场革命仍在迅速展开。该项目将测试CRISPR-CAS9如何区分正确和不正确的DNA目标的假设，并将一种称为定点自旋标记的生物物理技术的能力提高到在生理条件下一次询问一个生物分子的水平。预计这项研究将产生机械上的理解，这将有助于进一步开发基因组编辑工具，并推动技术进步，使更广泛的科学界受益。该项目还将通过研究生和本科一级的研究和课程开发，促进对科学、技术和经济研究人员的培训，并将支持就与基因组编辑有关的问题向公众进行外联，这些问题具有深刻的社会impacts.Clustered-Regularly-Interspaced-Short-Palindromic-Repeats(CRISPR)和CRISPR相关(CAS)蛋白质，可为细菌和古菌提供适应性免疫。在II型CRISPR中，单个Cas9蛋白被小RNA(S)激活，在特定位置切割DNA双链。Cas9靶获得的关键步骤是解开DNA双链，形成稳定的R环结构，其中RNA引导片段与DNA Protspacer的靶链碱基配对。研究已经建立了CAS9目标获取的顺序展开模型，这些机械性的见解有助于推动基于CRISPR的技术的发展。然而，对Cas9功能机制的了解还远远不够。在这个项目中，Aim 1将使用自旋标记和荧光光谱相结合的方法绘制Cas9诱导的DNA在本体溶液中解离的边界；Aim 2将研究Cas9靶切割与DNA解离边界变化之间的相互依赖关系；Aim 3将开发自旋标记方法，在单分子水平上研究Cas9诱导的DNA解离。这项工作将通过测试目标辨别的“两态平衡”假设，促进对CRISPR-CAS9功能的基本理解，并将产生新的生物物理技术，对核酸和蛋白质-核酸复合体的研究产生广泛影响。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。

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.