Tracing and harnessing the ancestry of RNA-guided nucleases

Objective

The phylogenetic, biochemical and functional diversity of these linked RGNs suggest a common origin that gave rise to the innumerable variations we observe today. However, little is known about these ancestors and the evolutionary paths that led to their modern counterparts. Recently, our team showed that reconstructed ancestors of Cas9 and Cas12a RGNs exhibit flexible substrate recognition and collateral activities distinct from any modern counterpart. This work sets the stage to uncover the evolutionary trajectories that gave rise to all related RGNs and to translate insights into new and improved biomolecular tools. As part of RGNcestry, we assembled four accomplished PIs covering CRISPR/transposon biology and technologies, ancestral sequence reconstruction and structural biology. The ensuing project will test our working hypothesis that ancestral RGNs consistently exhibit flexible properties that allow them to function in roles paralleling their modern counterparts, albeit less effectively due to a lack of specialization. To test this hypothesis and translate the resulting discoveries, we will pursue four objectives: 1. Reconstruct a detailed ancestral tree uniting RuvC-harboring RGNs. 2. Define the properties and functional roles of ancestral RGNs. 3. Explore molecular features and dynamics of ancestral RGNs. 4. Enhance and apply ancestral RGNs. If successful, our high-risk/high-reward project could reveal how RGNs evolved into the rich diversity observed today and establish a new paradigm for RGN engineering and application spanning genome editing to molecular diagnostics.