Mechanistic investigation of DNA cleavage and specificity in CRISPR-Cas9

CRISPR-Cas9 中 DNA 切割和特异性的机制研究

• 批准号: 1905374
• 负责人: Giulia Palermo
• 金额: $ 45万
• 依托单位: University of California-Riverside
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1905374&HistoricalAwards=false
• 关键词: Mechanistic; investigation; DNA; cleavage; specificity; Mechanistic; investigation; DNA; cleavage; specificity

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.

通过该奖项，化学过程的化学过程计划是为加利福尼亚大学河滨分校的Giulia Palermo博士提供资金，以调查CRISPR中DNA裂解和特异性的分子基础（群集定期散布的短palindromic重复序列）-CAS9系统通过计算方法。 CRISPR-CAS9技术是一种使生物体（包括人类）基因组进行精确修饰的方法。 该技术基于使用核酸酶，能够切割双链DNA的酶以及与酶结合的RNA分子，并将酶引导到将发生切割的DNA中的位点。 这项研究旨在了解酶使用Palermo博士及其合作者开发的计算方法功能的机制。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，生物燃料生产以及具有增强营养价值的抗旱作物的开发中的应用。该项目涉及一项宣传和指导计划，其中包括为来自代表性不足的少数群体和老师的高中生动手会议。Crispr-CAS9是一种细菌适应性免疫系统，通过启用便捷的基因组编辑技术来彻底改变基础和应用生命科学。该项目详细了解了该系统如何编辑和操纵核酸，这对于提高基因组编辑能力至关重要。 该研究项目旨在通过使用最先进的计算方法来表征DNA裂解和特异性的机制和特异性。该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）与自由能方法一起研究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，这对于DNA的酶促加工至关重要。经典的MD和增强的采样技术用于研究DNA特异性的机理，表征了因改变的DNA序列的结合以及它们如何影响催化而引起的构象变化。理论研究是与实验者合作进行的。 新理论有助于解释实验数据，并做出可以在实验室中进行测试的可能预测。该奖项反映了NSF的法定任务，并且使用基金会的知识分子优点和更广泛的影响审查标准，被认为是值得通过评估来支持的。