CAREER: Functional and evolutionary analysis of transposon-encoded, RNA-guided nucleases across the three domains

This project will investigate abundant genetic elements known as transposons, or ‘jumping genes,’ and will specifically examine a primordial family of transposon-encoded enzymes that use guide RNAs to target and cut matching DNA sequences. These enzymes are directly related to CRISPR-Cas9 and CRISPR-Cas12, which similarly rely on guide RNAs for targeted DNA cleavage, and which have revolutionized scientists’ ability to perform precise genetic modifications in plants, animals, and humans. By exploring what may be the evolutionary ancestors of these enzymes, this project has the potential to uncover new tools for genome engineering and new knowledge about diverse biological pathways where RNA-guided DNA targeting offers an advantage to host cells and genetic parasites. CRISPR-based tools continue to disrupt biotechnology sectors, posing urgent new questions about how to best harness these developments for societal benefit. Promoting broader public knowledge about CRISPR, genetics, and precision medicine will be of central importance in making informed decisions about how we shepherd CRISPR technology in the future. The broader impacts of this project include a commitment to educating middle- and high-school students in STEM on these topics, thereby inspiring the next generation of young thinkers to pursue careers in science and research. TnpB-family enzymes were recently shown to use transposon-encoded guide RNAs to direct cleavage of complementary DNA target sequences, but the biological and functional importance of this biochemical activity remains unclear. Even more mysteriously, TnpB-family genes are present across all three domains of life in diverse genetic contexts, and in association with distinct families of transposases. This project will investigate the hypothesis that that RNA-guided nucleases play a fundamental role in promoting transposon survival and proliferation, explaining their widespread conservation in bacteria, archaea, and eukarya. This problem will be studied across three major experimental aims, including the determination of how transposases and nucleases coordinate their enzymatic activities; the exploration of RNA-guided nucleases encoded by diverse bacterial transposons; and the discovery of novel nucleases encoded within archaeal and eukaryotic transposable elements. These aims will employ a multidisciplinary approach blending phylogenetics, biochemistry, genetics, and high-throughput sequencing. By elucidating the molecular origins of CRISPR RNA-guided nucleases, this project will fundamentally transform our knowledge and appreciation of transposon domestication during the evolution of host immune systems, and will offer new opportunities to develop next-generation genome editing tools. 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.