Mechanistic investigation of DNA cleavage and specificity in CRISPR-Cas9

有了这个奖项，化学部门的生命过程化学项目资助了加州大学河滨分校的Giulia Palermo博士，通过计算方法研究CRISPR（聚集规律间隔短回文重复）-Cas9系统中DNA切割的分子基础和特异性。CRISPR-Cas9技术是一种能够精确修改生物（包括人类）基因组的方法。这项技术是基于一种核酸酶的使用，一种能够切割双链DNA的酶，以及一种与酶结合并引导酶到达DNA中将要切割的位置的RNA分子。这项研究试图通过Palermo博士和她的合作者开发的计算方法来了解酶的功能机制。这项研究的结果可能有助于开发更有效的基因组编辑技术及其在生物学研究、生物燃料生产和开发具有更高营养价值的抗旱作物中的应用。该项目包括一项外联和指导计划，其中包括为来自未被充分代表的少数群体的高中生和教师提供实践课程。CRISPR-Cas9是一种细菌适应性免疫系统，通过实现简单的基因组编辑技术，正在彻底改变基础和应用生命科学。该项目提供了对该系统如何编辑和操纵核酸的详细了解，这对提高基因组编辑能力具有重要意义。本研究项目旨在利用最先进的计算方法表征化脓性链球菌（Sp） CRISPR-Cas9系统的DNA切割机制和特异性。本项目采用混合量子力学/分子力学（QM/MM）方法和ab-initio分子动力学（MD）模拟（使用Born-Oppenheimer和Car-Parrinello方法），结合自由能方法研究CRISPR-Cas9中DNA裂解的催化机制。这些方法可以阐明金属离子的催化作用，金属离子对DNA的酶处理至关重要。采用经典的MD和增强的采样技术来研究DNA特异性的机制，表征由改变的DNA序列结合引起的构象变化以及它们如何影响催化。理论研究是与实验人员合作进行的。这个新理论有助于解释实验数据，并做出可以在实验室中验证的可能预测。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。

With this award, the Chemistry of Life Processes Program in the Chemistry Division is funding Dr. Giulia Palermo from the University of California, Riverside, to investigate the molecular basis of DNA cleavage and specificity in the CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 system through computational methods. The CRISPR-Cas9 technology is a method that makes precise modification of the genome of an organism (including that of humans) possible. The technology is based on the use of a nuclease, an enzyme capable of cutting the double stranded DNA, and an RNA molecule that is bound to the enzyme and guides the enzyme to the site in the DNA where the cut is to take place. This research seeks to understand the mechanism by which the enzyme functions using computational methods developed by Dr. Palermo and her collaborators. The results of this study may aid in the development of more efficient genome editing technologies and their applications in biological research, biofuels production, and the development of drought-resistant crops with enhanced nutritional value. The project involves an outreach and mentoring program, which includes hands-on sessions for high school students from underrepresented minority groups and teachers.CRISPR-Cas9 is a bacterial adaptive immune system that is revolutionizing basic and applied life sciences by enabling a facile genome editing technology. This project provides detailed understanding of how this system edits and manipulates nucleic acids, which is of importance for improving the genome editing capability. This research project seeks to characterize the mechanism of DNA cleavage and specificity of the Streptococcus Pyogenes (Sp) CRISPR-Cas9 system by using state-of-the-art computational methods. The project employs a mixed quantum mechanics/molecular mechanics (QM/MM) approach and ab-initio Molecular Dynamics (MD) simulations (using the Born-Oppenheimer and Car-Parrinello approaches) in combination with free energy methods to investigate the catalytic mechanism of DNA cleavage in CRISPR-Cas9. These methodologies may elucidate the catalytic role of metal ions, which are critical for the enzymatic processing of DNA. Classical MD and enhanced sampling techniques are employed to investigate the mechanism of DNA specificity, characterizing the conformational changes arising from the binding of altered DNA sequences and how they affect the catalysis. Theoretical investigations are performed in collaboration with experimentalists. The new theory assists in the interpretation of experimental data and makes possible predictions that can be tested in the laboratory.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.