University of South Carolina Aiken aiken, South Carolina
Body of Abstract: DNA transposable elements are segments of DNA that are mobilized by transposase proteins. They are found in virtually all eukaryotic genomes and their presence often affects nearby gene expression. The 430 base pair mPing transposable element from rice is derived from the larger, autonomous Pingelement. mPing is currently being developed into a targeted insertion tool for genome editing. Previous experiments have shown that mPingexcised by transposase can be inserted into Cas9 cleaved sites by non-homologous end joining. This method would be improved if we can create a version of mPing that has normal excision but with reduced capacity for insertion, preventing off target insertion. Thus, our goal is to further understand mPing’sinsertion mechanism. Mobilization of mPing is induced by binding of transposase and ORF1 proteins to the ends of the element and catalyzing staggered cleavage of the 3 base pair target site duplication (TSD) on either side of the element. Previous studies have found that altering the TSD from TTA or TAA can decrease mPing excision depending on which base is changed, but the effect on insertion is unknown. We are measuring insertion of the mmPing20:URA3 element with altered TSD sequences by performing yeast transposition assays and testing if the resulting ADE2revertant colonies can grow on plates with FOA or lacking uracil. These experiments should help to determine the role these sequences play in transposase mediated insertion.