Structural and evolutionary basis for insertion unidirectionality in RNA-guided DNA transposition systems

PROJECT SUMMARY CRISPR-associated transposons (CAST) represent a class of recently discovered bacterial genetic elements that can perform programmable transposition. Using a user-provided RNA sequence, CASTs can insert kilobases of DNA into specific sites in the genome, a crucial function that cannot be achieved with current state of the art genome editing tools. Thus, CASTs match an unmet need in therapeutics for genetic disorders plagued by gene mutations like cystic fibrosis. To optimize CASTs for application in next-generation genome editing tools, a comprehensive understanding of how it inserts DNA is required. Of particular interest is its unidirectionality, referring to CAST’s propensity to insert DNA in a single orientation. While unidirectionality is a coveted feature of CASTs, its mechanism remains uncharacterized. Components implied to contribute to this mechanism are the signature ends flanking the element. These ends recruit proteins, TnsB in model system shCAST, that excise the transposon from its original location to prepare it for insertion elsewhere. However, these ends are different in sequence, described in this proposal as “asymmetric”, suggesting that TnsB performs different functions at each end that could be used to achieve unidirectionality. This project proposes an interdisciplinary project that will identify key drivers for unidirectionality. Specific aim 1 will use high-resolution cryo-electron microscopy (cryo-EM) to structurally visualize the shCAST paired-end complex, a stage of transposition involving interactions between the asymmetric ends facilitated by TnsB. This structure will identify key features that distinguish the ends from one another from a 3-D standpoint. Follow-up characterization of the transpososome, the next stage of transposition involving the assembly of the paired-end complex with other CAST proteins, will recognize how the identified features convey unidirectionality. Specific aim 2 will utilize bioinformatics to identify features conserved across all CAST systems that establish orientation specificity. Metagenomic mining will scan the vast trove of sequence data to compile a comprehensive list of CAST elements. This expansive set enables evolutionary analysis of CASTs to determine global trends maintaining unidirectionality and will define each element’s mechanism based on retention of conserved features. Biochemical assays will be used to validate features identified in either aim. The PI will be extensively trained in both cryo-EM and bioinformatics skills along with critical thinking to ensure seamless integration of experimental and computational results. The Kellogg and Feschotte labs at Cornell University have access to a wide-ranging set of resources that ensure mastery of these skills and will also emphasize soft skills like communication to ensure the PI’s development as a well-rounded scientist. Together, this proposal sets forth a plan to comprehensively understand CAST unidirectionality and its associated mechanisms, enabling its optimization and implementation in genome editing tools of interest.