Peer review of: CRISPR GUARD protects off-target sites from Cas9 nuclease activity using short guide RNAs (2020)

 
Reviewer #1: 
Remarks to the Author: 
In this manuscript, the authors have designed a series of sgRNAs designed to reduce off-target 
gene editing by Cas9. The authors explore a series of RNA designs, characterizing the reduction of 
off-target editing rates both in test tubes as well as cell lines. 
 
The paper had several strengths, which are listed below. 
 
(1) The paper was written clearly. 
 
(2) The figures, in general, were clear and easy to interpret. 
 
(3) The authors did not overstate their results, or the implications thereof. 
 
(4) I particularly enjoyed Sup Figure 3; whether reducing off-target 1 increased off-target 2-N was 
a very clever question to ask / answer. 
 
There were also several aspects that should be improved before publication, which are also listed 
below. 
 
(A) In Figure 2d and 2f, the experiment should be repeated with additional bioreps. It is difficult to 
interpret the data with the number of bioreps, and the resultant error bars. 
 
(B) Please consider adding additional text to clarify what is plotted in Figure 2E. I was able to 
interpret it, but general readers with less experience in the field may have difficulty. 
 
(C) This is a minor comment, and therefore isn't critical, but I would refrain from using the word 
'damage' when describing editing events (line 131). 
 
(D) I understand the need for pre-complexing the target sgRNA with active Cas9 and the GUARD 
RNA with the dCas9. It is, for this experimental setup, the appropriate combination. However, it 
would be good for the authors to discuss the inherent complexity of using this approach in the real 
world at the end of the paper. 
 
(E) The authors correctly describe the potential risk for guide swapping. When the on-target event 
is mediated by any dCas9 variant (e.g., activation, base editing, etc.), even the shortened GUARD 
RNAs, if they bind to their own DNA, should still result in activation, base editing, etc., if the RNAs 
are swapped. If I am mistaken, please clarify more in the text. If I am correct, I would perform an 
additional experiment to address this issue. 
 
 
 
Reviewer #2: 
Remarks to the Author: 
This paper describes a simple and useful method for suppressing off-target genome edits by 
delivering GUARD RNAs, which are truncated gRNAs having perfect complementarity to the 
potential off-target sites. Multiplexing was possible to block multiple off-target edits 
simultaneously, and the authors suggest some GUARD RNA design rules even though it should be 
elaborated further. Finally, the authors have shown that base editors work even with truncated 
gRNAs, which makes GUARD RNA less useful for suppressing off-target base editing. However, 
when carefully designed to avoid cytosine at the GUARD RNA binding sites, the method would be a 
valuable tool for base editing as well. 
 
Overall, new findings and usefulness of the method make this paper suitable for publication in 

Nature Communications. However, there are several issues that should be addressed. 
 
1. One needs to identify potential off-target sites before starting the genome editing, and it is not 
trivial. As a solution, ‘GUARDfinder’ has been developed, but in many cases, there may be so 
many potential off-target sites, which in turn make it extremely difficult to screen all potential 
GUARD RNAs. The authors should describe these shortcomings and suggest potential solutions at 
the discussion part. 
 
2. Some concepts are confusing, so they need to be described more clearly. In addition, there are 
many mistakes in the manuscript. 
 
(Line 61) For the ‘15-nt + spacer’ GUARD RNA, how did the authors design the 5 nt spacer 
sequence? 
 
(Figure 2) It seems like that the same set of GUARD RNA was used for on-target and off-target 
cleavage in Figure 2c. In figure 2d, however, two types of GUARD RNAs was used (14 nt VEGFA 
GUARD and 14 nt CAVIN4 GUARD), and it is not clear what ‘VEGFA GUARD’ is. This should be 
explained clearly in the manuscript. Otherwise, the left panel of the figure 2d should show the data 
using CAVIN4 GUARD just as in figure 2c. 
 
(Line 573) Information about statistics in figure 2f is not shown. 
 
(Line 661) It is about figure 2e, not 2d. 
 
In figure 2f and figure S2, it seem like that the same set of on-target and off-target sites were 
used, but the labels on the figures are different, which makes the manuscript confusing. In 
addition, it is not shown that which type of GUARD RNA was used in figure S2. 
 
(Figure 3b) It would be better if the authors could show the directionality of gRNAs and GUARD 
RNAS more clearly (e.g. by indicating termini as 5’ or 3’). Figure S6b seems clearer, so those kinds 
of supplementary figures could be added. 
 
(Figure 4a) How was the % base editing calculated? Because there are several cytosines at the 
editing window, the way of calculation should be clearly described. 
 
There are many mistakes in the manuscript when assigning the figure 5. 
 
In figure 6b, the plots are showing % base editing, but in the figure legend, it is written as indel 
and SNP from BE. Clearer description is required. 
 
In figure 6d, it should be described clearly what kind of measurements were compared for 
calculating P values. 
 
In generating plots, the same axis labels should be used throughout the manuscript. For example, 
some plots have the label ‘Base editing’ while some have ‘BE’. 
 
3. In Figure S3, the authors investigated CAVIN4 as the off-target site for the plasmid-based 
genome editing experiments, but investigated the other off-targets sites for the RNP-based assay. 
Is there any reason for this? It would be better it the authors could show off-target effects in 
CAVIN4 to be able to compare the plasmid- and RNP-based delivery. 
 
4. Sometimes, it is hard to read labels in the figure. Resolution and font size should be increased. 
 
5. In table 1, are the data from GUARD RNA transfection showing the reads at the on-target 
(EMX1) amplicon or corresponding off-target regions? 

 
6. In figure 4b, it seems like that HDLBPhiGC GUARD RNA did not decrease off-target base editing 
at the gRNA editing window while it did in the case of indel formation (Figure S4) at the same 
gRNA:GUARD RNA ratio, but gRNA-editable cytosines may not be open by the GUARD RNA 
considering the sequence context. Is there any explanation for this? 
 
7. Figure 6f can be modified to give more information, not only to describe the factors affecting 
GUARD RNA performance, but also to suggest some design guidelines the authors have identified. 
 
8. It would be good if the authors could show examples of using ‘GUARDfinder’ as a supplementary 
figure.