The activity of the mammalian DNA transposon piggyBat from Myotis lucifugus is restricted by its own terminal inverted repeat. The activity of the mammalian DNA transposon piggyBat from Myotis lucifugus is restricted by its own terminal inverted repeat

The transposon piggyBat has already been used as a genomic tool for studies in human cells13. Here, through a combination of in vitro DNase I footprinting experiments with cell culture-based transposition assays, we have been able to rationalize the organization of the piggyBat TIRs. Based on this, we solved the cryo-EM structure of the piggyBat pre-synaptic complex at 3.4Å resolution. By combining these results, we discovered that piggyBat's transposition activity in vivo is severely restricted by a transposase binding site on its Left End (LE). Further modifications of the RE and the elimination of N-terminal phosphorylation sites of the transposase and the duplication of C-terminal site-specific DNA domain increased transposition activity by approximately two orders of magnitude relative to wild type. Taken together, the results indicate a previously unobserved down-regulation of transposon activity by a elongated TIR. The TIR modifications described here lead to a transposition activity increase comparable to the most highly active reported piggyBac version with no detectable change in chromosomal integration fidelity Overall design: To determine the transposition profiles of WT and pBat-4StoA-2xCRD (mutant) transposases genomewide, we established HTC116 cells expressing WT or mutant transposases and carrying a Puromycin-resistance encoding self-reporting transposon cassette carrying a Puromycin-resistance gene. We then performed selective RT-PCR amplification of transcripts carrying the left end (LE) of the pBAT transposon from Puromycin-resistant cells, followed by RNAseq analysis to characterize and compare the transposition insertion site profiles generated by WT and mutant transposases across the HTC116 genome.