Peer review of: Synergistic gene editing in human iPS cells via cell cycle and DNA repair modulation (2020)

 

 

Reviewers' Comments: 
 
Reviewer #1: 
Remarks to the Author: 
Summary: 
The authors have submitted a very commendable rebuttal to the Referee comments from their original 
submission, with satisfactory responses to most concerns. Most importantly, the authors have added 
additional data on endogenous sites, which was requested by all referees. Their results across 5/8 new 
endogenous sites aren’t as impressive as the improvements in HDR rates observed across the original 
2 sites, but overall the trend appears to constitute a meaningful advance. Pending that the authors 
address the remaining comments below, this Referee recommends publication. 
 
Remaining comments: 
 
1. In Figures 6g and 6h, 5/10 sites exhibit low levels of HDR (lower total editing + much lower HDR 
compared to the 5 other sites). Can the authors identify something about these sites, or the targeting 
strategy (distance of cut site from SNP) that would segregate these two groups of results? 
2. Similar to comment #1, the effect of the cocktail appears to be most pronounced for sites/gRNAs 
with higher basal HDR. To make this point more clear, the authors should include a fold-change 
summary plots of the data in Figures 6g and 6h (dots for each site representing fold-change) so that 
readers can get a sense of the expected mean / range of improvement with no treatment, XL+N+S, 
and 32C+N+S. 
3. Why do the authors observe a dichotomy of superior performance of XL+N+S or 32C+N+S 
depending on the site (split roughly evenly)? 
4. Several Figure references in the rebuttal appear to refer to incorrect panels, making it difficult to 
follow the author’s logic in their response. 
 
 
 
Reviewer #2: 
Remarks to the Author: 
In the submitted manuscript, the authors described approaches to enhance HDR-mediated precise 
genome editing in pluripotent stem cells. The current work is a much improved version of their 
previous manuscript submitted to Nature Biotechnology. My biggest concern then was that the authors 
based their conclusions primarily on a traffic light reporter and that there was minimal validation on 
endogenous genomic loci. Hence, I am appreciative that the authors have tried to address this 
concern with additional experiments on a handful of endogenous target sites. Overall, the data look 
reasonably convincing. Below are some comments that I have for improving the authors' work. 
 
1) Figure 4e: Why do the "-" (untreated control) and "DMSO" results look so different? The graph 
seems to suggest that the editing outcomes can be altered simply by adding DMSO (~12% vs ~30% 
HDR). 
 

2) Figure 4g: Can the authors show DMSO, NU7441, and SCR7 in the graph as well? 
 
3) Figure 6g-h: Ideally, there should be error bars with p-values to indicate whether any improvement 
is significant. 
 
Best wishes, 
Meng How Tan 
 
 
 
Reviewer #3: 
Remarks to the Author: 
Highly efficient precise gene editing in stem cells remains a challenge. This manuscript describes a 
useful method to quantify HDR versus NHEJ in single cells during gene editing. Using a fluorescent 
reporter system, the authors can measure conversion of GFP to BFP using single cell FACS analysis to 
quantify the percentage of HDR versus NHEJ. Using different oligo donors, the manuscript show 
efficient derivation of monoallelic mutants using a number of technical improvements. The method will 
be a useful resource for precise gene editing. The observation of synergistic effects in the last figure is 
interesting. 
 
Major comments: 
 
1. The manuscript presents a number of technical advances, including a fluorescent reporter system, 
cell cycle control, modulating HDR/NHEJ pathways using small molecules, and cold shock conditions. I 
suggest they provide a final summary figure to system summarize what they learned, which is helpful 
for HDR in iPS cells and which is synergistic with other conditions. While several of these conditions 
are derived from prior works, a summary is helpful to the broad audience understand major 
observations from this work. 
 
2. There are six main figures. Some of the figures can be moved to supplemental to keep the 
manuscript concise and easy to read. 
 
3. I am interested in learning whether the conditions also altered cell growth, morphology, and 
viability. As a useful method, this information should be provided to readers for their evaluation. 
 
4. The citation style is not for Nat Comm.