Mechanistic investigation of DNA cleavage and specificity in CRISPR-Cas9

凭借这一奖项，化学部的生命过程化学计划资助加州大学河滨分校的Giulia Palermo博士通过计算方法研究CRISPR(簇状规则间隔短回文重复序列)-Cas9系统中DNA切割和特异性的分子基础。CRISPR-Cas9技术是一种使生物体(包括人类)基因组的精确修改成为可能的方法。这项技术的基础是使用核酸酶，一种能够切割双链DNA的酶，以及一种与酶结合并将酶引导到DNA中发生切割的位置的RNA分子。这项研究试图利用巴勒莫博士和她的合作者开发的计算方法来了解这种酶的作用机制。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，并将其应用于生物研究、生物燃料生产以及开发具有更高营养价值的抗旱作物。该项目包括一个外展和指导计划，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是一个细菌适应性免疫系统，它通过启用一种简单的基因组编辑技术来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，这对于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来表征化脓性链球菌(Sp)CRISPR-Cas9系统的DNA切割机制和特异性。该项目采用量子力学/分子力学(QM/MM)混合方法和从头计算分子动力学(MD)模拟(使用Born-Oppenheimer和Car-Parrinello方法)，并结合自由能方法来研究CRISPR-Cas9中DNA切割的催化机理。这些方法可能会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。经典的分子动力学和增强的采样技术被用来研究DNA特异性的机制，表征由于改变的DNA序列的结合而引起的构象变化以及它们如何影响催化作用。理论研究是与实验者合作进行的。这一新理论有助于解释实验数据，并做出可在实验室测试的预测。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势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.