CAREER: Mechanistic Investigations of Conformational Activation and Catalysis in Emerging CRISPR-Cas Systems

职业：新兴 CRISPR-Cas 系统中构象激活和催化的机制研究

• 批准号: 2144823
• 负责人: Giulia Palermo
• 金额: $ 65.01万
• 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: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2027-08-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2144823&HistoricalAwards=false
• 关键词: CAREER; Mechanistic; Investigations; Conformational; Activation

This award is funded in whole or in part under the American Rescue Plan Act of 2021 (Public Law 117-2).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 mechanisms underlying the catalytic activities of newly emerging CRISPR (clustered regularly interspaced short palindromic repeat)-Cas systems. Inspired by the initial CRISPR-Cas9 gene editing tool, several novel CRISPR-Cas systems have recently been developed that precisely manipulate DNA and RNA in a cell. State-of-the-art of computational methods will be used to determine how these new systems change the chemical composition of DNA bases, cut both strands of the DNA double-helix, and target RNA. The results from this study are expected to aid in the development of novel CRISPR-Cas-based biotechnologies, such as nucleic acid detection and imaging. In addition, the applications of methods development aspects of this project have the potential to extend from the biosciences to fuel production and to the development of drought-resistant crops. This project will integrate with an educational plan that mentors a large cohort of pre-college and undergraduate students, including women and those from underrepresented minority groups. Students will receive their first hands-on experience in program coding and computation, building the technical skills to succeed in the 21st century technological workplace. CRISPR-Cas is an integral part of a bacterial adaptive immune response that has revolutionized life sciences as a genome editing tool. This project will employ advanced computational methods to characterize the mechanisms of conformational activation and catalysis in novel CRISPR-Cas systems, including Cas12a and Cas13a and emerging Cas9-based complexes. Long timescale molecular dynamics (MD) simulations will be combined with a variety of enhanced sampling techniques and free-energy methods to thoroughly investigate the conformational changes of the Cas proteins that prompt nucleic acid cleavages. Mixed quantum mechanics/molecular mechanics (QM/MM) approaches and ab-initio MD will be used to elucidate details of the chemical mechanisms driving the catalysis of nucleic acids, with the potential to enhance catalytic efficiency and guide enzyme design. The computational studies will be coordinated in collaboration with experimentalists to assist with the interpretation of experimental data and enable testable predictions. Overall, this study will help elucidate the biomolecular function of these emergent CRISPR-Cas systems, bridging the gap between understanding molecular mechanisms and engineering applications.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.

该奖项全部或部分由2021年美国救援计划法案资助（公法117-2）。有了这个奖项，化学系的生命过程化学项目正在资助来自滨江的加州大学的朱利亚巴勒莫博士，研究新出现的CRISPR催化活性的分子机制（成簇的规则间隔的短回文重复）-Cas系统。受最初的CRISPR-Cas9基因编辑工具的启发，最近开发了几种新型CRISPR-Cas系统，可以精确操纵细胞中的DNA和RNA。最先进的计算方法将用于确定这些新系统如何改变DNA碱基的化学组成，切割DNA双螺旋的两条链，以及靶向RNA。这项研究的结果有望有助于开发基于CRISPR-Cas的新型生物技术，如核酸检测和成像。此外，该项目方法开发方面的应用有可能从生物科学扩展到燃料生产和抗旱作物的开发。该项目将与一项教育计划相结合，该计划指导了一大批大学预科和本科生，包括妇女和来自代表性不足的少数群体的学生。学生将获得他们在程序编码和计算方面的第一次实践经验，建立在21世纪世纪技术工作场所取得成功的技术技能。CRISPR-Cas是细菌适应性免疫反应的一个组成部分，它作为基因组编辑工具彻底改变了生命科学。该项目将采用先进的计算方法来表征新型CRISPR-Cas系统中的构象激活和催化机制，包括Cas 12 a和Cas 13 a以及新兴的基于Cas9的复合物。长时间尺度的分子动力学（MD）模拟将与各种增强的采样技术和自由能方法相结合，以彻底研究促使核酸裂解的Cas蛋白的构象变化。混合量子力学/分子力学（QM/MM）方法和从头算MD将用于阐明驱动核酸催化的化学机制的细节，具有提高催化效率和指导酶设计的潜力。计算研究将与实验学家合作协调，以协助解释实验数据并实现可检验的预测。总的来说，这项研究将有助于阐明这些新兴CRISPR-Cas系统的生物分子功能，弥合理解分子机制和工程应用之间的差距。该奖项反映了NSF的法定使命，并被认为值得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来支持。

项目成果

Enhancement of CRISPR/Cas12a trans-cleavage activity using hairpin DNA reporters.

• DOI: 10.1093/nar/gkac578
• 发表时间: 2022-08-12
• 期刊: NUCLEIC ACIDS RESEARCH
• 影响因子: 14.9
• 作者: Rossetti, Marianna;Merlo, Rosa;Bagheri, Neda;Moscone, Danila;Valenti, Anna;Saha, Aakash;Arantes, Pablo R.;Ippodrino, Rudy;Ricci, Francesco;Treglia, Ida;Delibato, Elisabetta;van der Oost, John;Palermo, Giulia;Perugino, Giuseppe;Porchetta, Alessandro
• 通讯作者: Porchetta, Alessandro

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

The electronic structure of genome editors from the first principles

从第一原理看基因组编辑器的电子结构

• DOI: 10.1088/2516-1075/acb410
• 发表时间: 2023
• 期刊: Electronic Structure
• 影响因子: 2.6
• 作者: Nierzwicki, Łukasz;Ahsan, Mohd;Palermo, Giulia
• 通讯作者: Palermo, Giulia