Peer review of: Prediction-based highly sensitive CRISPR off-target validation using target-specific DNA (2019)

Reviewers' Comments:
Reviewer #1:
Remarks to the Author:
The authors attempt to apply the CUT-PCR method towards detection of off-target genome editing.
By treating the edited genomic DNA with nucleases designed to deplete unmodified, wildtype,
sequences and subsequent PCR amplification cycles, even tiny amounts of modified genomic DNA
could be detected by high-throughput sequencing.
This work represents an innovative application of an existing technology towards an important field
that complements current off-target detection methods. Due to its novelty, ease of use, and
extremely high sensitivity, I recommend publication of this work in Nature Communications.
However, I feel that the pros and cons of the technology have not been sufficiently described and
demonstrated, which reduces the impact of the publication.
Major Concern:
1. As the authors have already alluded to many times, it seems clear and logical that this approach
will more efficiently enrich deletions rather than insertions, due to the bias in PCR (smaller
fragments are amplified better). In fact, Supplementary Figure 6a shows that insertions that are
detected in non-amplified sample are undetectable after amplification. The strengths and
weaknesses of this technology will be better illuminated if the authors discuss the effect of
insertion mutants and deletion mutants before and after amplification. Perhaps several sites that
are known to generate both deletion and insertion mutations can be investigated to ascertain the
degree of enrichment and de-enrichment the two types of mutations undergo.
Other concerns:
1. Related to above, the authors state that they observe more enrichment of larger deletion
products than deletions containing fewer bases (1-2), and correctly state that mutants with one
base deletion may be a substrate during CRISPR enrichment. In fact, it seems that deletions below
1 or 2 bases undergo de-enrichment (Figure 1d, Supplementary Figure 4a,b,c,d,e, Supplementary
Figure 5a,b,c, Supplementary Figure 6a). There are some on or off target sites where CRISPR
nucleases only generate predominantly 1 base deletion or insertion (Nature 563, 646–651 (2018))
Would the technology fail to enrich (or detect) such sites?
2. The authors rely on the presence of a Cpf1 PAM in the editing window of adenine base editor so
that ABE activity abolishes the Cpf1 PAM site. The authors should clearly state that this is a pre-
requisite for ABE off-target enrichment. Also, this will only enrich off-target mutants that have
base edits at the PAM region of the enrichment guide. The authors should clearly state that
mutants that generate adenine editing at other sites in the region will not be enriched. How could
one enrich sites that do not have a suitable A/T-rich region that could be used as a Cpf1 PAM?
 3. Would the use of high-fidelity versions of CRISPR enzymes reduce the bias towards different
 indel products during enrichment? More generally, would the choice of CRISPR nuclease during the
 enrichment affect the result (degree and sensitivity of the enrichment)?
 
Reviewer #2:
Remarks to the Author:
In this manuscript, Kang et al. developed a CRISPR amplification method to amplify and detect
small amounts of off-target mutations induced by CRISPR effectors. Two key factors of the method
are accurate in-silico prediction of potential off-target sites and removal of background wild-type
DNA. In this study, the authors compared CRISPR amplification with conventional NGS or GUIDE-
seq for the detection of off-target mutations and found that extremely small amounts of off-target
 mutations below the NGS detection level could be detected by CRISPR amplification, and the
 sensitivity was augmented with increasing amplification cycles. This study is of potential interest
 and some suggestions are listed below to revise this study.
Major
1) The major finding of this study is that the CRISPR amplification method can be used to detect
extremely small amounts of off-target mutations below the NGS detection level. Though the
experiments generally support the enrichment properties of this method, the sensitivity and
necessity of this method need to be tested at more sites. In this manuscript, off-target mutations
at most sites can still be detected by other methods, so the necessity of CRISPR amplification
cannot be supported sufficiently.
2) The NC data shown in Fig. 2a, 3a and 4a indicated that multiple amplification would induce false
positive results and therefore interfere the accuracy of the method.
3) The authors suggested that “mutated DNA with larger indels is relatively better amplified”,
which is inconsistent with the data shown in Fig. S4a-d, S5, S6a. The authors should provide more
evidence to support their claim.
 Minor
 1) The editing window of ABE shown in Fig. 4c is confusing. The editing window of ABE has been
 shown to be positions 4-9 (Gaudelli et al., 2018, Nature, PMID: 29160308) but the authors
 showed 12-18 in the manuscript. In addition, the authors did not mention which version of ABE
 they used in their study.