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

CRISPR-Cas9系统是一种开创性的工具，可以改变包括人类在内的各种生物体的DNA，并在治疗学、农业和其他领域具有巨大的潜力。为了确保安全和有效的应用，需要更高的精度和效率。CRISPR-Cas12系统及其类似物由于其天然的丰富性和多样性，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。这个项目的主要目标是探索CRISPR-Cas12内切酶及其类似物的工作机制，以促进它们作为改进的基因组编辑工具的发展。同时，该项目包括一个教育部分，以丰富普渡大学学生、K-12学生和普通公众等不同受众对CRISPR技术的了解。这将通过春节外展计划和暑期研究计划中的学生动手培训来实现。此外，该项目将有助于公众教育，促进社区对CRISPR技术的好处和风险的讨论，并丰富各级学生的学习经验。拟议的研究将通过揭开共存的抗CRISPR蛋白之间被忽视的调控相互作用，并阐明新兴但未被探索的Cas12类似物的结构和机制，来解决CRISPR领域的两个关键知识空白。该项目将利用结构生物学和生物化学的尖端方法，研究被称为抗CRISPR蛋白的天然抑制蛋白的调控机制(AIM 1)，并剖析在可移动遗传元件和病毒中发现的新发现的Cas12类似物的结构动力学，如TnpB和Fanzor(Aim 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。该奖项由生物科学总监分子和细胞生物科学部的分子生物物理学计划资助。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.