Uncovering the molecular function of novel transposon-encoded Cas9 homologs

PROJECT SUMMARY Mobile genetic elements (MGEs) possess the ability to mobilize within genomes and/or between genomes from distinct species, and are a major driving force in the spread of antibiotic resistance and virulence genes. Due to the unrelenting assault of MGEs, prokaryotic organisms have evolved numerous sophisticated defense systems that operate on both an innate and adaptive level, and in some cases directly target MGE nucleic acids in a sequence-specific manner. Of notable importance in recent years are immune systems encoded by clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) genes, which employ guide RNAs for the targeted binding and cleavage of foreign nucleic acids. Remarkably, Cas genes themselves have evolved from genes encoded within MGEs, and transposons in particular. A striking example is the RNA-guided DNA endonuclease, Cas9, which evolved directly from a distinct group of homologous transposon-encoded proteins within the TnpB family (and is hereafter referred to as Cas9H), whose molecular functions are entirely unknown. Beyond these initial bioinformatic observations, my recent analyses have uncovered a conserved non-coding RNA (ncRNA) that shows strong genetic association with Cas9H genes. I hypothesize that ancient, transposon-encoded Cas9 homologs function together with guide-like RNAs, to modulate the excision, efficiency, and/or target site specificity during transposition of the mobile element. By discovering this primordial biological function, my work will offer insights into the evolutionary trajectory of CRISPR-Cas9 and uncover core enzymatic properties that nature used as starting material to arrive at a potent and highly programmable RNA-guided DNA endonuclease. In Aim 1, I will bioinformatically identify transposable elements containing Cas9 homologs to prioritize candidates for experimental study, and develop a robust heterologous expression system to monitor transposition events. Importantly by monitoring genome-wide insertion specificity will inform whether Cas9H modulates target selection. In Aim 2, I will elucidate the function of the non-coding ‘HEARO’ RNA through systematic mutagenesis, and determine the role of Cas9H nuclease domains in transposition. Finally, in Aim 3, I will adopt a biochemical approach to directly probe protein-RNA interactions between Cas9H and the ncRNA, and uncover the role of its conserved nuclease domains, laying a foundation for future structural studies that may shed light on ancient scaffolds conserved between TnpB and Cas9. This project will leverage my training as a molecular geneticist and expand my abilities in bioinformatics and biochemistry. These experiments will be the first to rigorously probe the function of a TnpB family protein, and reveal for the first time how TnpB/Cas9H modulates transposition. In addition to providing insights into the evolutionary origins of CRISPR-Cas immune systems, completion of this work will also inform the development of new genetic engineering tools.