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 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.