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.

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.受到最初的CRISPR-CAS9基因编辑工具的启发，最近已经开发出了几种新型的CRISPR-CAS系统，这些系统精确地操纵了细胞中的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双螺旋的两个链和靶RNA。这项研究的结果预计有助于开发基于CRISPR-CAS的新型生物技术，例如核酸检测和成像。此外，该项目的开发方面的应用有可能从生物科学延伸到燃料生产和抗旱作物的发展。该项目将与一项教育计划集成，该计划指导大量的学前班和本科生，包括妇女和代表人数不足的少数群体。学生将获得他们在计划编码和计算方面的第一个实践经验，建立在21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不可或缺的一部分，它彻底改变了生命科学作为基因组编辑工具。该项目将采用先进的计算方法来表征新型CRISPR-CAS系统中构象激活和催化的机制，包括CAS12A和CAS13A以及基于CAS9的复合物。长时间分子动力学（MD）模拟将与多种增强的采样技术和自由能方法结合使用，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AB-Initio MD将用于阐明驱动核酸催化的化学机制的细节，以提高催化效率并指导酶设计。计算研究将与实验者合作协调，以协助解释实验数据并启用可测试的预测。总体而言，这项研究将有助于阐明这些新兴的CRISPR-CAS系统的生物分子功能，从而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的法定任务，并被认为是值得通过基金会的知识分子优点和更广泛影响的审查标准来通过评估来支持的。

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