Project Summary Recently, new macromolecular systems have been discovered which marries the benefits of both CRISPR and TNP systems and shows tremendous promise as programmable DNA-insertion tools for genome-editing, complementing the power of tools such as CRISPR-Cas9. This proposal aims to uncover the molecular mechanisms governing two as-yet poorly understood phenomena in CRISPR-Transposase (CRISPR-TNP) systems: target-site immunity and programmed-DNA insertion. The central protein thought to be responsible for both of these observed behaviors in the multi-component shCAST system is shTnsC. We propose to utilize high- resolution cryo-EM to determine the structure of DNA-bound shTnsC. In addition, shTnsC is a AAA+ ATPase whose nucleotide-hydrolysis activity is linked to transposition. We propose to determine the structure of shTnsC in different nucleotide-bound states in order to reveal the role of ATP-hydrolysis in transposition. Finally, we aim to uncover the mechanisms governing shTnsC recruitment to the target-site via Cas12k and shTniQ, and how the association between these factors ultimately initiates shCAST transposition. Our strong preliminary data indicates that these aims are likely to be successful. In addition, the Kellogg lab is well-supported within the Cornell community to achieve the goals outlined in this proposal.