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

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.

有了这个奖项，化学部的生命过程化学项目正在资助来自滨江加州大学的Giulia巴勒莫博士，通过计算方法研究CRISPR（成簇的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技术是一种可以精确修饰生物体（包括人类）基因组的方法。 该技术基于使用核酸酶，一种能够切割双链DNA的酶，以及与酶结合并将酶引导至DNA中发生切割的位点的RNA分子。 这项研究旨在了解酶的功能机制，使用巴勒莫博士和她的合作者开发的计算方法。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研究，生物燃料生产和开发具有增强营养价值的抗旱作物中的应用。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。 该研究项目旨在通过使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。该项目采用混合量子力学/分子力学（QM/MM）方法和从头算分子动力学（MD）模拟（使用Born-Oppenheimer和Car-Parrinello方法）结合自由能方法来研究CRISPR-Cas9中DNA切割的催化机制。这些方法可以阐明金属离子的催化作用，这是至关重要的DNA的酶促加工。经典分子动力学和增强采样技术被用来研究DNA特异性的机制，表征由改变的DNA序列的结合所产生的构象变化以及它们如何影响催化作用。理论研究是与实验学家合作进行的。 新的理论有助于解释实验数据，并做出可以在实验室测试的预测。该奖项反映了NSF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。

项目成果

Factor H-Inspired Design of Peptide Biomarkers of the Complement C3d Protein

H 因子启发的补体 C3d 蛋白肽生物标志物的设计

• DOI: 10.1021/acsmedchemlett.9b00663
• 发表时间: 2020
• 期刊: ACS Medicinal Chemistry Letters
• 影响因子: 4.2
• 作者: Harrison, Reed E.;Zewde, Nehemiah T.;Narkhede, Yogesh B.;Hsu, Rohaine V.;Morikis, Dimitrios;Vullev, Valentine I.;Palermo, Giulia
• 通讯作者: Palermo, Giulia

Spontaneous Embedding of DNA Mismatches Within the RNA:DNA Hybrid of CRISPR-Cas9

• DOI: 10.3389/fmolb.2020.00039
• 发表时间: 2020-03-17
• 期刊: FRONTIERS IN MOLECULAR BIOSCIENCES
• 影响因子: 5
• 作者: Mitchell, Brandon P.;Hsu, Rohaine, V;Palermo, Giulia
• 通讯作者: Palermo, Giulia

Establishing the fundamental rules for genetic code expansion

建立遗传密码扩展的基本规则

• DOI: 10.1038/s41557-023-01243-9
• 发表时间: 2023
• 期刊: Nature Chemistry
• 影响因子: 21.8
• 作者: Sinha, Souvik;Ahsan, Mohd;Palermo, Giulia
• 通讯作者: Palermo, Giulia

Structural basis for Cas9 off-target activity.

CAS9非目标活动的结构基础。

• DOI: 10.1016/j.cell.2022.09.026
• 发表时间: 2022-10-27
• 期刊: Cell
• 影响因子: 64.5
• 作者: Pacesa M;Lin CH;Cléry A;Saha A;Arantes PR;Bargsten K;Irby MJ;Allain FH;Palermo G;Cameron P;Donohoue PD;Jinek M
• 通讯作者: Jinek M

Enhanced specificity mutations perturb allosteric signaling in CRISPR-Cas9.

• DOI: 10.7554/elife.73601
• 发表时间: 2021-12-15
• 期刊: eLife
• 影响因子: 7.7
• 作者: Nierzwicki L;East KW;Morzan UN;Arantes PR;Batista VS;Lisi GP;Palermo G
• 通讯作者: Palermo G