Peer review of: Structural basis of seamless excision and specific targeting by piggyBac transposase (2020)

REVIEWERS' COMMENTS: 

Reviewer #1 (Remarks to the Author): 

This manuscript presents the cryoEM structures of piggyBAC transpososomes in two different reaction 
states although with supporting biochemistry. 
The structures are very intriguing and add new features and insights to the transpososome zoo. As the 
authors point out, one aspect of piggyBAC that is useful in biotech is that it leaves its previous host-
DNA location with 4bp sticky ends that can be simply ligated together, leaving no trace that the 
transposon was ever there at all, and without requiring error-prone repair processes. This is quite 
unusual among transposases and the structure reveals how it does this. Additionally, the authors were 
able to design new transposon ends that not only support their hypotheses but also enhance activity. 

Although the biochemistry appears to be fine and is not the primary “new knowledge” of the paper, it 
would be helpful if it were described and discussed more clearly. 

Specific points, mostly aimed at clarity not substance: 

Is the density clear enough to support all the H-bonds shown in Figures 4d and e and 5? 

For all the biochemistry figures (including the supplementary ones), the legends are excessively 
concise. Please add concentrations of proteins and DNAs, and a bit more information in general. 

The structural work is described in refreshingly useful detail. 

Supplementary figure 1b: Please label the lanes and label which cartoon pertains to which lane. It 
took some time to figure out that the cartoon between gels refers to the right lane of each gel rather 
than a different experiment run on the right gel. 

Figure s1c and d: it would be nice to see all the variations on the ends tested side-by-side in the same 
assay rather than the different assays used in parts c and d. 
I can’t find the methods for the experiment in Figure s1d, and the diagram is puzzling: if pUC was 
linearized by XmaI digest, what are the nicks at each end? If this is an ethidium-stained gel then why 
do some DNA ends have little stars on them? 
The only activity I can see is in lane 3, so why does the legend specify that little activity was seen in 
lane 2 when there’s no decrease in substrate in lane 1 either? 
What is the schmutz in the bottom of lane 1? 
If only 4 sites in pUC19 are used as targets as the 2nd paragraph of the methods states, why so many 
bands in lane 3? 

The product-sequencing is a minimally-consequential part of this paper - the authors should be 
praised for completeness in doing it even though it is simply confirms they see with their transposon 
ends what other groups have seen before for piggyBAC. That said, it could be better-described and 
better documented (it is essentially all “data not shown”). 
The 1st paragraph of “results” implies that what was sequenced was the products of LE35 
oligonucleotide insertion, but the 2nd paragraph of the methods section says that for sequencing a 
completely different in vivo transposition reaction was done using LE35 plus RE63 on the two ends of 
the (sub-minimally described) mini-transposon. (that paragraph could also use a topic sentence: one 
doesn’t know what experiment is being described until the end). 

P7, bottom – It makes sense that the target-binding channel narrows between the hairpin and STC 
structures, but could it also be that the channel is simply flexible, and other classes of particles that 
were discarded during processing had other widths? 
P8, middle – worth noting that the there is an A-tract between the two CRD-binding sites, which would 
naturally tend to narrow the minor groove (if I remember correctly, 5’A-3’T steps don’t disrupt A-tract 
structures). In Figure 3b it seems to adopt a cannonical A-tract structure with propeller twist as well 
as narrow minor groove – is that true? 

Figure 4d vs. the text describing it on the top of p10 puzzle me sorely. I can’t find Y439 in the figure, 
although Y406 is shown (blue over blue, hard to see). In the view shown, Y283 doesn’t seem to be 
near anything interesting. I also don’t see the other residues involved in the methyl-binding pocket. It 
might be worth making a closer-up version of d and e? It might lose context but the important details 
might show better. 

In figure 4 the number schemes for target vs. hairpin nucleotides are confusing, both schemes conflict 
with figure 4g, and neither are explained until figure 5. 

Figure 4f is really cool and worth a little more weight in the text! 

The mode of target recognition is very interesting and surprising. 

P12: please give a bit more explanation of the “colony count” assay – even if it is previously 
published, one shouldn’t have to read the reference list to figure out the basics of what’s going on 
here. Both the figure legends and methods are rather cryptic. What PCR primers were used? What is 
pTpB? What is the significance of resistance to G418? Etc. Also, the figure would be a little more at-a-
glance comprehensible if the 2nd panels were labeled “insertion” rather than “colony count”. Or is the 
“colony count” assay looking at complete transposition from one location to another? Why do the 
excision vs. colony count assays give somewhat different answers? Or are both panels measuring 
excision only (from a G418-resistance gene)? 

P12, last paragraph: I don’t understand the argument about how the fact RE 1-33 is functional in vitro 
supports the formation of tetramers in vivo. Yes, the part colored black could in a pinch still act as a 
binding site for the CRD – but wouldn’t that just make a dimeric complex exactly as shown in your 
nice EM structures? The first sentence on p12 implies that simply adding more protein might enhance 
activity (although the phrase “away from the transposon tip and its site of catalysis” is awkwardly 
placed) – was that tried? 

Is there any evidence for contacts between the CRDs and the rest of the transposase as hinted at in 
figure 6c? Such contacts wouldn’t be necessary for the model, but without them I wouldn’t call it a 
“tetramer” – just two dimers. 

Figure 6e is very clever and potentially quite useful. 

Typos, etc: 
Abstract, near end: “synaptic formation” should be synaptic complex formation” 
Figures 2c and b are swapped. 
P11, 1st paragraph: “with which R281 and Y291 interact …” is a misplaced modifier. 
P12, 1st full paragraph – “dimeric” should be “dimer” 
P14, bottom: “recognition and integration is” should be “… are”, and I think “in vitro” should be in 
front of “reactions involving hairpin formation …” 

Reviewer #2 (Remarks to the Author): 

The PiggyBac (PB) transposon is a prominent genome engineering tool used in transgenesis, genetic 
screening, stem cell biology and gene therapy. Given its applied significance, a mechanistic 
understanding of the PB transposition pathway has been long sought. In this manuscript, Chen et al. 
present the first structural views of PB. Using cryoEM, the authors describe two structures of the PB 
transposase: one in complex with excised transposon end DNA (SNHP) and one with the transposon 
ends inserted into the cognate genomic target sequence (STC). Unexpectedly, the structures reveal an 
asymmetric molecular arrangement, which helps explain PBs unique features. Using cleverly designed 
in vitro and in vivo transposition assays, the authors support the notion that structural asymmetry 
promotes transposon end recognition and synapsis, supporting PBs activity in cells. Furthermore, the 
work elucidates the structural principles of PB’s target site selection and sheds new light onto 
transposase hyperactivity. 
Overall, this study presents important insights into a process of high biotechnological and medical 
relevance. The experiments were carefully designed and conducted, and the paper is very well written 
and easy to follow. Undoubtedly, the presented results will be of broad interest; they will likely provide 
inspiration for studying diverse transposons and for improving their use in genome engineering. Thus, 
I only have a few minor questions and suggestions, which the authors may wish to consider. 

- In the abstract: “The results show that the structure of the excision intermediate and the precision of 
TTAA targeting create the link that give rise to the specific properties of piggyBac.” It is not easy to 
appreciate the essence of this sentence for the uninformed reader, please revise. 
- Second paragraph of the introduction: The term “genome editing” is predominantly used when 
changes are made on a site-directed manner (e.g. in the context of CRISPR/Cas-, ZFN- or TALEN-
mediated modifications). As SB- and PB-mediated modifications occur throughout the genome, 
alternative terms - such as genome engineering or gene insertion - seem more appropriate. 
- Did the SNHP structure provide any insights into cleavage site selection on the NTS? Why does the 
3’OH of the TS attack the NTS exactly 4nts into the flanking DNA, and how does it come there? Can 
the mechanism be similar to what was seem for Hermes and RAG? Also, does the structure reveal why 
the size of the hairpin tip differs in Tn5 and PB? 
- Page 10, last paragraph: As the two strands of the TTAA target sequence are located on separate 
DNA molecules, it is not immediately obvious why the authors have expected them to pair. Perhaps it 
will be worthwhile to refer to STC complex structures of Mu and Mos1, where the same DNA design 
yielded base paired target sites to clarify this point. 
- On a related point, the authors suggest that TTAA is already melted in the target DNA prior to strand 
transfer. Do the structures provide any clues for what can drive such distortions in dsDNA? 
- Page 11, 2nd paragraph: “This unusual mode of transposon target selection is in line with our 
inability to identify any part of PB that would recognize the TTAA target in dsDNA form.” Please clarify 
if this comment refers to structure analysis or experimental data. 
- Regarding the in vivo transposition assays, can the authors clarify why additional CRD binding sites 
in LE increase PB activity? 
- On page 14, the discussion regarding structural coupling of the transposition steps in PB and other 
systems is interesting, but could perhaps be extended a bit. How are the steps of PB more linked than 
of other elements and how exactly is this connected to seamless excision? Perhaps contrasting to a 
different element could be helpful. If I understand correctly, PB’s main trick is to excise through a 4 nt 
hairpin intermediate, exactly embracing its palindromic TSD, which leaves complementary overhangs 
on the flanks for seamless repair. 
- Can the authors speculate why hyPBase is less dependent on transposon end asymmetry?