Peer review of: Frataxin gene editing rescues Friedreich’s ataxia pathology in dorsal root ganglia organoid- (2020)

REVIEWERS' COMMENTS:
Reviewer #1 (Remarks to the Author):
Reviewer #2 (Remarks to the Author):
 The majority of the concerns have been addressed satisfactory and the overall quality over the
 work has improved.
The issues raised about the previous version of this paper have largely been addressed in this
revision. Two minor points:
1. The paragraph in the introduction about mechanisms of epigenetic suppression of FXN
expression needs some improvement. There is no clear link between non-B structures formed by
long GAA repeats in vitro and epigenetic mechanisms, so their role in vivo remains hypothetical.
Epigenetic mechanisms include repressive (rather than abnormal) histone marks such as loss of
acetylation and increased di-trimethylation at both H3K9 and H3K27, formation of D-loops, and
DNA methylation. As these changes are explored in the cell model described in the paper, they
should more clearly summarized in the introduction. Also, animal models (e.g. YG8 mice) in
addition to cell models and patient samples have shown these changes.
2. The statements that that “2D iPSC-SNs form unorganized clusters in a random pattern with
unproven participation of different neuron subtypes, fail to recapitulate the DRGs spatial
architecture and cellular diversity” and “when we tried to differentiate FRDA patient iPSCs into
iPSC-SNs, an extensive cell death occurred such that we were unable to obtain neuronal cultures
of decent quality with enough double PRPH+/BRN3A+ sensory neurons on which to perform
studies” should be tempered in the light of several reports of SN differentiation from iPSCs,
including from FRDA patients, which turned out to be viable and of controlled composition (e.g. Lai
et al. JBC 2019; 294:1846-1859 and most recently Dionisi et al. Sci Rep 2020; 10:7752). This
does not affect the importance, interest, power and elegance of the 3D model developed by the
authors, even if 2D models are also effective.
Reviewer #3 (Remarks to the Author):
 The revised version of the manuscript is much improved and the authors have answered to most
 of the major and minor comments. The addition of the scRNA sequencing of the control DRGO
 brings a wealth of information.
 There are still a few minor comments that should be addressed:
1. The molecular characterization of the FRDA DRGO is very preliminary, and would benefit from
further investigation before making statements such “Thus, these results demonstrate that
diseased DRGOs recapitulated many peculiar aspects of the pathological phenotype present in
patient somatic cells. Furthermore, the response to oxidative stress in both the mitochondrial and
cytoplasmic compartments and the impairment in the generation of Fe-S proteins...”. From the
data presented, there is no evidence of impairment of Fe-S cluster biogenesis. The deficiency of
aconitase appears to be at the transcriptional level and is most likely a reflection of the decrease
biogenesis of mitochondria. It is understandable that the authors might consider the deep
characterization of the molecular phenotype as a story beyond the current manuscript (and this is
comprehensible). However, the strong affirmative statements in the results and to a lesser extend
in the discussion should be revised to reflect the results obtained.
2. There are a few mistakes in the figures (e.g. legend of Fig.S1 does not correspond to what is
seen in Figure p33 line 1078, no * on Fig. 3b)
3. There are still a few typos throughout the manuscript (e.g. p11, line 354, p 30 line 974)