CAREER: Expanding and Leveraging the Natural Diversity of Type I CRISPR for Genome Engineering

CRISPR-Cas are widespread microbial immune systems against parasitic foreign DNA. They also gave rise to the Nobel Prize-winning Cas9 gene-editing tools that have revolutionized the ability to install programmable genetic modifications in plants and animals. This project will investigate CRISPR-Cas3 (Type I CRISPR-Cas system), which is the most abundant and phylogenetically diverse CRISPR branch found in nature. By exploiting unique features of Type I CRISPR unavailable to Cas9, this project has the potential to develop new technologies and applications with unprecedented targeting flexibility, which will significantly advance human genome research. Furthermore, new biological insights will be generated about the diversity of prokaryotic genetic interference pathways. As CRISPR technologies continue to transform agriculture, medicine, and biotechnology sectors, enabling broad public understanding of the scientific and societal impacts is an important undertaking. This project includes public outreach through CRISPR-themed museum exhibitions and high school educational programs, to empower the next generation of young thinkers to pursue careers in science and technology. The enzymatic machinery of Type I CRISPR has many molecular properties unavailable to Cas9 but is under-developed in biotechnology. The overall research goal of this study is to advance the fundamental understanding of divergent Type I CRISPR-Cas systems and broaden their utilities in human genome engineering. This project will pursue two research objectives, including (1) the development of existing Type I CRISPR variants into novel gene-editing platforms to expand the targeting scope and applications of the current CRISPR toolkit; and (2) the expansion of Type I CRISPR’s natural diversity by establishing brand-new classes of Type I effectors and elucidating the mechanisms whereby they mediate bacterial defense. A multifaceted approach blending human cell culture, high-throughput sequencing, and bacterial genetics will be employed. This work has the potential to transform our understanding of the diversity, function, and biotechnological potential of multi-subunit CRISPR-Cas systems. This project is supported by the Genetic Mechanisms program and the Systems and Synthetic Biology program of the Division of Molecular and Cellular Biosciences in the Directorate for Biological Sciences. 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.