University of South Carolina Aiken Swansea, South Carolina
Body of Abstract: Transposable elements (TEs) are segments of DNA that when mobilized by specific transposase proteins, can excise, and reinsert in the genome. The mPing TE is highly active in rice and has been used as an activation tag to upregulate the expression of nearby genes. Programable nucleases such as Cas9 have been successfully used to induce small mutations or deletions in plant genomes, but sequence insertion by homologous recombination has been less efficient. Recent experiments have shown that linking Pong Transposase (TPase) and Cas9 together allows for insertion of mPing into specific genomic locations. Our goal is to develop a yeast assay for quickly testing Cas9-mediated targeted insertion of mPing. We first used our established yeast transposition assay to test how TPase function is affected by fusion to the Cas9 protein. We observed that the fusion proteins show a significant loss in transposition, but that the addition of longer linkers slightly increases transposition frequency. To test the effect of TPase on Cas9 function, we induced transposition in the presence or absence of a guide RNA targeting CAN1. Screening our ADE2 revertant colonies for canavanine resistance showed that canavanine resistance was produced by the TPase:Cas9 fusion protein only in the presence of the guide RNA. This suggests that the Cas9 protein is not inhibited by the presence of TPase. To test for targeted insertion of mPing in yeast, we are developing a novel transposition reporter plasmid and mPing element that will allow for expression of the ADE2 gene upon targeted insertion.