Peer review of: Genome-wide specificity of dCpf1 cytidine base editors (2023)

Reviewers' Comments: 
 
Reviewer #1: 
Remarks to the Author: 
In the present study, Kim and colleagues intended to assess specificity of dCpf1 base editor, an 
alternative deamination-mediated base editing system that requires distinct PAM sequence and 
does not involve DNA nicking. By modifying their Digenome-seq method, which is based on in vitro 
digestion of isolated genome followed by WGS, the authors tried to provide a comprehensive assay 
to detect genome-wide deaminated sites. The authors argued that their method is highly sensitive 
and detects distinct off-target candidates from the ones of the nuclease version. However, as this 
method seems to detect many false positives and depends on the mismatch criteria to narrow 
down the number of candidate sites, the reviewer is skeptical of how much their modified 
Digenome-seq contributes to improve the detection of unexpected off-targets. Besides, the 
present study cannot address the recent issue of gRNA-independent off-targets for cytosine base 
editors. The findings that the off-targets for dCpf1 base editor are partly distinct from Cpf1, and 
that the Apobec1 variants reduces gRNA-dependent off-target, are not quite surprising. Therefore, 
this reviewer cannot recommend this manuscript to be published in Nature Communications. 
 
Major comments 
1. The authors need to mention and estimate the false positive rate of their method and perform 
comparison with pure prediction-based detection. It will be also beneficial to score off-target 
candidates based on their Digenome-seq result and compare them with actual off-target rates in 
the cells. 
 
2. If ecomonically and technically acceptable, false positives could be reduced by increasing the 
depth of the reads, using other NGS system with longer reads, or enrichment of SSB samples by 
devising size selection (i.e. producing larger fragments at shearing, followed by end repair and size 
selection for smaller fragments). 
 
3. The authors need to assess if USER treatment alone may cause significant SSB. 
 
4. The readers would be more interested if the authors could discuss if dCpf1 base editor is 
superior in its specificity, compared to conventional SpCas9-based one. 
 
Minor comments 
 
L128-129: “A total of 123,309 SSB sites were captured at the DYRK1A site.” 
Is this supposed to mean in the entire genome? Even so, this huge number suggests that this 
assay captures too much false positives probably due to technical and statistical limitation. The 
filtering based on the mismatch criteria cannot solve this statistical problem and thus the false 
positives remain. 
 
L143-144: “Note that the average number of in vitro LbCpf1 cleavage sites was 6 ± 3, which is 
fewer than that of dLbCpf1-BE.” 
As the detection method for LbCpf1 nuclease off-target is different and perhaps more accurate, it 
may not be fair to compare these numbers without considering false positives. 
 
L164-165: “The 20 validated off-target sites exhibited base editing frequencies that ranged from 
0.1% to 22.4%.” 
Considering the detection limit of amplicon-seq by the NGS system, is it really possible to detect 
0.1% SNV with statistical significance? 
 
 
 
Reviewer #2: 
Remarks to the Author: 
The manuscript “Genome-wide specificity of dCpf1 cytidine base editors” describes off-target 
activity of dCpf1-BE ( C-to-U base editor fused to catalytically inactive dCpf1) in comparison to 
Cpf1 nuclease. Targeted amplicon sequencing reveals that the dCpf1 base editor is more tolerant 
to mismatches that the Cpf1 nuclease. The authors describe a variation of Digenome-seq, a 
method that has been developed in the same lab and used to identify genome-wide off targets of 
RNA-guided nucleases (Cas9 and Cpf1) as well as Cas9-nickases fused to base editors. The original 
method is based on detecting double strand breaks in vitro. Here the authors modified Digenome-
seq to identify genome-wide off-targets detecting single strand breaks of a C-to-U base editor 
fused to catalytically inactive dCpf1 (dCpf1-BE) in vitro. Genome-wide C-to-U conversion are then 
identified by a unique alignment pattern observed by whole genome sequencing (WGS). More 
genome-wide off-targets are observed for dCpf1-BE when compared to that of Cpf1 nuclease. Off-
targets identified in vitro are then tested using targeted sequencing in HEK293 cells showing that 
the base editor and nuclease recognize different off-target sites. 
 
Overall, this is a good study expanding on the previously developed digenome-seq method 
comparing off-target differences between the dCpf1 base editor and Cpf1 nuclease. Although the 
experimental work is convincing and worth publishing, the manuscript in its current form can be 
confusing in places and lacks depth in data description. Data need to be presented more clearly. In 
addition the discussion should be expanded to put findings in this study in perspective with 
literature on off-targets by nCas9-base editors using SNV analysis. 
 
Major comments: 
 
The manuscript lacks description of statistical analysis used. For example, are three-fold 
differences in off-target activity between dCpf1-BE and Cpf1 statistically significant. Or were the 
authors considering significant changes only when there was also a three-fold difference. Statistics 
used should be added to figure legends and method section. This applies to several figures, eg. 
Fig1, Sup Fig2, etc… 
 
All analysis of modified Digenome-seq is focused on sites that have some resemblance with the 
target region. This does minimize the power of this whole genome method especially since 
genome-wide off-target effects have been reported for BE3 that DO NOT correlate with similarity 
to the target site (PMID:30819928). By focusing on sequencing similarity, the number of SSB 
(single strand breaks) observed for DYRK1A gets reduced from 123,309 SSBs to ~20 potential off-
targets. This does not sound like an unbiased genome-wide approach. I feel strongly that if 
Digenome-seq is a good unbiased method, there should be more information that can be extracted 
from 123,309 SSBs. Focused analysis on sequence similarity should only be a follow-up analysis. 
 
Are Digenome-seq results from a single replicate? This needs to be clearly stated. Would the most 
of the 123,309 SSBs be the same in biological replicates? Would they then be relevant? Biological 
replicates would be the best way to determine “real” off-targets. Did I miss that information? 
 
I understand that there could be limitation to this method as it is performed in vitro. Possible 
advantages and limitations of Digenome-seq should be addressed in the discussion of the 
manuscript and compared to findings of relevant literature (PMID:30819928, PMID:30995674, …) 
 
Paragraph “Genome-wide off-target sites of dLbCpf1-BE revealed by Digenome-seq” 
This paragraph needs a lot more detail. The numbers do not make any sense. I cannot evaluate 
this paragraph in its current form. How many genes were tested? Where do 106 target sites come 
from? I tried to do the math, but still cannot figure it out. Please add all information to the results 
section in the main text. 
Line 151: “across three other target sites (Fig 3a)”. This is not correct. 
Line 151: “8 out of 59 cleavage sites”. Where do they come from? Which genes? List # of sites per 
target gene 

Fig3c: The sequence logo was determined for off-targets, but is not described in results section. 
Sequence motif analysis should also be shown for DYRK1A in Fig3c and for CDKN2A and EMX1 in 
Supp Fig 3. Findings should be summarized in the results section and interpretation added to the 
discussion. 
 
Fig 3d/e: Please add base editing efficiency of dCpf1-BE in an additional column in the table. It is 
hard to compare between table and bar graph. 
 
The results of Cpf1 variants tested in this manuscript are disappointing. However, the description 
of variants is vague and should be described in more detail. A comparison to other tested Cpf1 
variants (PMID:30742127, PMID:30995674) would be helpful. Were any of the mutations similar 
or in similar regions? 
 
Minor comments: 
 
Line 27: Please specify range of SSBs rather than an average of 12 SSBs 
 
Line 61: Please state clearly at beginning of first paragraph that targeted amplicon sequencing was 
used to determine indels 
 
Line 62: It is hard to follow what the different targets without looking at the Supplemetal Fig 1a. It 
would be helpful to take the time to list the nine target sites in the results paragraph. 
 
Line 62: human genomic needs space 
 
Suppl Fig 1 and Fig 2: What is the difference between DNMT1, EMX1 and DNMT1-3 and EMX1-2? 
Differences re-occur eg in Fig 3. Be consistent if it is they are the same or explain if they are 
different. Please adjust all affected nomenclature in text and figures.