CAREER: Probing structural dynamics and regulatory mechanisms of RNA-guided CRISPR-Cas12 endonucleases and their analogues

职业：探索 RNA 引导的 CRISPR-Cas12 核酸内切酶及其类似物的结构动力学和调控机制

• 批准号: 2339799
• 负责人: Leifu Chang
• 金额: $ 129.66万
• 依托单位: Purdue University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2024
• 资助国家: 美国
• 起止时间: 2024-01-01 至 2028-12-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2339799&HistoricalAwards=false
• 关键词: CAREER; Probing; structural; dynamics; regulatory

The CRISPR-Cas9 system is a groundbreaking tool for altering DNA in a wide range of organisms, including humans and holds immense potential for therapeutics, agriculture, and beyond. To ensure safe and effective applications, greater precision and efficiency is needed. The CRISPR-Cas12 system and analogues, due to their natural abundance and diversity, potentially offer key advantages over Cas9 in terms of specificity, efficiency, and adaptability to different genomic contexts. The primary goal of this project is to explore the working mechanism of CRISPR-Cas12 endonucleases and their analogues, which could facilitate their development as improved genome editing tools. In parallel, the project includes an educational component to enrich the understanding of CRISPR technology among a diverse audience, including students at Purdue University, K-12 students, and the general public. This will be achieved through a Spring Fest outreach program and hands on training of students in a summer research program. In addition, this project will contribute to public education, foster community discussions about the benefits and risks of CRISPR technology and enrich learning experiences for students at all levels.The proposed research will address two critical knowledge gaps in the CRISPR field by unraveling the overlooked regulatory interplay among coexisting anti-CRISPR proteins and elucidating the structure and mechanisms of emerging yet underexplored Cas12 analogues. Employing the cutting-edge methodologies of structural biology and biochemistry, the project will investigate the regulatory mechanism of natural inhibitor proteins, known as anti-CRISPR proteins (Aim 1), and dissect the structural dynamics of newly identified Cas12 analogues found in mobile genetic elements and viruses, such as TnpB and Fanzor (Aim 2). Collectively, this project will expand knowledge in genome editing and promote wider community awareness and appreciation of CRISPR technology.This award is funded by the Molecular Biophysics Program in the Molecular and Cellular Biosciences Division of the Biological Sciences Directorate.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.

CRISPR-Cas9 系统是一种突破性的工具，可改变包括人类在内的多种生物体的 DNA，在治疗、农业等领域具有巨大的潜力。为了确保安全有效的应用，需要更高的精度和效率。 CRISPR-Cas12 系统和类似物由于其天然丰度和多样性，在特异性、效率和对不同基因组环境的适应性方面可能比 Cas9 具有关键优势。该项目的主要目标是探索 CRISPR-Cas12 核酸内切酶及其类似物的工作机制，这可以促进它们作为改进的基因组编辑工具的发展。与此同时，该项目还包括一个教育部分，旨在丰富不同受众（包括普渡大学学生、K-12 学生和公众）对 CRISPR 技术的理解。这将通过春季节外展计划和夏季研究计划中的学生实践培训来实现。此外，该项目还将促进公共教育，促进关于 CRISPR 技术的好处和风险的社区讨论，并丰富各级学生的学习经验。拟议的研究将通过阐明被忽视的监管问题来解决 CRISPR 领域的两个关键知识差距共存的抗 CRISPR 蛋白之间的相互作用，并阐明新兴但尚未充分探索的 Cas12 类似物的结构和机制。 该项目将采用结构生物学和生物化学的尖端方法，研究天然抑制蛋白（称为抗 CRISPR 蛋白）的调节机制（目标 1），并剖析在移动遗传元件中发现的新鉴定的 Cas12 类似物的结构动力学和病毒，例如 TnpB 和 Fanzor（目标 2）。总的来说，该项目将扩展基因组编辑方面的知识，并促进更广泛的社区对 CRISPR 技术的认识和欣赏。该奖项由生物科学理事会分子和细胞生物科学部的分子生物物理学项目资助。该奖项反映了 NSF 的法定使命和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，该项目被认为值得支持。