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

Purdue University, West Lafayette IN

Investigators

Abstract

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.