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Abstract Modern plant breeding increasingly relies on genomic information to guide crop improvement. Although some genes are characterized, additional tools are needed to effectively identify and characterize genes associated with crop traits. To address this need, themPingelement from rice was modified to serve as an activation tag to induce expression of nearby genes. Embedding promoter sequences inmPingresulted in a decrease in overall transposition rate; however, this effect was negated by using a hyperactive version ofmPingcalledmmPing20. Transgenic soybean events carryingmPing‐based activation tags and the appropriate transposase expression cassettes showed evidence of transposition. Expression analysis of a line that contained a heritable insertion of themmPing20Factivation tag indicated that the activation tag induced overexpression of the nearby soybean genes. This represents a significant advance in gene discovery technology as activation tags have the potential to induce more phenotypes than the originalmPingelement, improving the overall effectiveness of the mutagenesis system. 

Introduction: Class II DNA transposable elements account for significant portions of eukaryotic genomes and contribute to genome evolution through their mobilization. To escape inactivating mutations and persist in the host genome over evolutionary time, these elements must be mobilized enough to result in additional copies. These elements utilize a “cut and paste” transposition mechanism that does not intrinsically include replication. However, elements such as the rice derived mPing element have been observed to increase in copy number over time. Methods: We used yeast transposition assays to test several parameters that could affect the excision and insertion of mPing and its related elements. This included development of novel strategies for measuring element insertion and sequencing insertion sites. Results: Increased transposase protein expression increased the mobilization frequency of a small (430 bp) element, while overexpression inhibition was observed for a larger (7,126 bp) element. Smaller element size increased both the frequency of excision and insertion of these elements. The effect of yeast ploidy on element excision, insertion, and copy number provided evidence that homology dependent repair allows for replicative transposition. These elements were found to preferentially insert into yeast rDNA repeat sequences. Discussion: Identifying the parameters that influence transposition of these elements will facilitate their use for gene discovery and genome editing. These insights in to the behavior of these elements also provide important clues into how class II transposable elements have shaped eukaryotic genomes. 

Class II DNA Transposable Elements (TEs) are moved from one location to another in the genome by the action of transposase proteins that bind to repeat sequences at the ends of the elements. Although the location TE insertion is mostly random, the addition of DNA binding domains to the transposase proteins has allowed for targeted insertion of some elements. In this study, the Gal4 binding domain was added to the transposase proteins, ORF1 and TPase, which mobilize the mPing element from rice. The Gal4:TPase construct was capable of increasing the number of mPing insertions into the Gal2 and Gal4 promoter sequences in yeast. While this confirms that mPing insertion preference can be manipulated, the target specificity is relatively low. Thus, the CRISPR/Cas9 system was tested for its ability to generate targeted insertion of mPing. A dCas9:TPase fusion protein had a low transposition rate suggesting that the addition of this large protein disrupts TPase function. Unfortunately, the use of a MS2 binding domain to localize the TPase to the MS2 hairpin containing gRNA failed to produce targeted insertion. Thus, our results suggest that the addition of small DNA binding domain to the N-terminal of TPase is the best strategy for targeted insertion of mPing.