High-Resolution Melting (HRM) Analysis for Screening Edited Lines: A Case Study in Vitis spp.

Round 1

Reviewer 1 Report

I have carefully reviewed this manuscript and found that it should be significantly revised. 
1. Novelty of the study should be indicated. 
2. Number of biological replications should be mentioned for all the parameters. 
3. Please improve the discussion by doing a compare and contrast of your findings with previous studies. 

Dear Authors,

After a careful review of your manuscript, I would like to share some constructive comments and suggestions that may help strengthen your work:

 

Abstract:

1. Simplify sentence structure and ensure tense consistency.
2. Qualify the claim about off-target detection with HRM.
3. Clarify the biological rationale for the target genes.
4. Explain “partially effective” in more detail.
5. Highlight the novelty of the approach compared to prior HRM applications.
6. Fix minor grammar, terminology, and redundancy.

 

Introduction:

1. The introduction is very dense, with long blocks of text and sentences that pack multiple concepts together (e.g., lines 43–55). Breaking them into shorter, focused sentences would improve readability.
2. The authors describe SNVs and indels but skip base editing and prime editing, which are also widely used. If the focus is only on CRISPR/Cas9-induced indels, they should make this scope explicit.
3. The discussion of European Commission’s GMO legislation (lines 63–71) is unusual in a scientific introduction. While it may be relevant, it disrupts the scientific flow and feels more suited to a discussion section or a brief contextual note. If kept, it should be shorter and directly tied to the study’s significance for grapevine research.
4. The research gap is not clearly articulated. The text says protoplast editing lacks a selection step and detection of small indels is challenging, but the specific unmet need is buried.

 

Materials and methods:

1. The sgRNA naming is inconsistent: (2) is repeated twice (lines 117–119).
2. Conditions for callus induction and Agrobacterium infection are only referenced, not described. At least a brief summary (e.g., media composition, selection agent concentration) should be included for reproducibility.
3. Only Nanodrop quantification is mentioned (line 147), which may not be accurate for purity. Fluorometric quantification (e.g., Qubit) would be more reliable.
4. Primer sequences are listed inline; better presentation would be a table of primers with expected product sizes.
5. The HRM assay mentions only “two technical replicates per sample” (line 171). For reliability, biological replicates should also be specified.
6. The HRM ramp (line 176) is described, but more details (e.g., data acquisition frequency, number of readings per °C increment) would improve reproducibility.
7. The description of PCA is unnecessarily mathematical (lines 204–211) for a methods section. A concise description of how PCA was applied to HRM data would suffice, with citations for statistical background.
8. The workflow for curve normalization (lines 184–191) should include justification for the chosen 80% confidence threshold.

 

Results:

1. Many passages (e.g., lines 321–337, 356–361) restate figure legends in full. This makes the results section redundant and harder to follow. Figure legends should carry descriptive detail; the main text should focus on interpretation.
2. In 3.1, only n=2 samples are tested per genotype (lines 263–264). This is too low to establish robustness of HRM specificity. The authors should include more biological replicates or at least acknowledge this limitation.
3. For greenhouse and in vitro plants (3.2), the number of biological and technical replicates is unclear.
4. The PCA descriptions are overly detailed in the text (lines 286–297, 303–317), while the figures themselves are complex. A better strategy is to summarize main findings in text and leave specifics to figure legends and supplements.

 

Discussion:

1. Discuss whether Sanger/NGS was used (or should be used) to confirm HRM-based classification.
2. Reframe HRM’s role as a preliminary screening tool that reduces the number of samples requiring costly NGS validation.
3. Highlight how HRM could accelerate grapevine editing pipelines.

 

Conclusion:

1. Clarify novel contributions of this study.
2. State limitations more explicitly.
3. Add future research/applications.
4. Refine claims about PCA to avoid overstatement.
5. Link findings to grapevine breeding relevance.
6. Improve clarity and conciseness of wording.

Author Response

3. Point-by-point response to Comments and Suggestions for Authors   Section Abstract:

Comments 1: Simplify sentence structure and ensure tense consistency.

Response 1: Done

Comments 2: Qualify the claim about off-target detection with HRM.

Response 2: The editing at the potential “off-target” site in VvEPFL9-2 was assessed, and no mutations were found in any of the transgenic lines (please see Fig. S4).

Comments 3. Clarify the biological rationale for the target genes.

Response 3. The two paralogous genes, VviEPFL9-1 and VviEPFL9-2, play a role in the induction of stomata cell fate. Shahbaz et al. (2025) functionally characterized these genes in grapevine and observed that, while both paralogous genes contribute to the establishment of stomata in leaf primordia, only VvEPFL9-2 is crucial for determining stomata density in growing leaves, since only its knock-out resulted in reduced stomata density in fully expanded leaves. This gene therefore has great potential to improve resilience to water stress. In fact, it has been demonstrated that grapevines edited in EPFL9-2 do not exhibit a significant impact on photosynthetic CO₂ absorption (Asat), with a 60% reduction in stomata having no effect on photosynthesis rates, but significantly improving water use efficiency.  

Comments 4. Explain “partially effective” in more detail.

Response 4. As described in the Results section, for some edited lines exhibiting a diverse range of mutations, the HRM analysis was unable to precisely determine the mutation profile or detect multiple patterns within a single sample. Nevertheless, it demonstrated a reasonable degree of reliability when used for screening purposes.

Comments 5. Highlight the novelty of the approach compared to prior HRM applications.

Response 5. A principal component analysis (PCA) was performed to explore the potential of dissociation curves in distinguishing among mutation profiles

Comments 6. Fix minor grammar, terminology, and redundancy.

Response 6. Done

Section Introduction

Comments 7. The introduction is very dense, with long blocks of text and sentences that pack multiple concepts together (e.g., lines 43–55). Breaking them into shorter, focused sentences would improve readability.

Response 7. Agree, done (line 39-55).

Comments 8. The authors describe SNVs and indels but skip base editing and prime editing, which are also widely used. If the focus is only on CRISPR/Cas9-induced indels, they should make this scope explicit.

Response 8. This is correct; however, we observed that in the edited lines obtained, most of the introduced mutations were indels. For this reason, we focused on this aspect in the text.

Comments 9. The discussion of European Commission’s GMO legislation (lines 63–71) is unusual in a scientific introduction. While it may be relevant, it disrupts the scientific flow and feels more suited to a discussion section or a brief contextual note. If kept, it should be shorter and directly tied to the study’s significance for grapevine research.

Response 9. The discussion regarding the European Commission’s legislation on edited plants has been included in the introduction to provide a comprehensive overview of the context and to explain the need for analytical tools for screening edited plants.

Comments 10. The research gap is not clearly articulated. The text says protoplast editing lacks a selection step and detection of small indels is challenging, but the specific unmet need is buried.

 

Response 10. Thank you for your comment. To clarify this point, we have revised the text to explicitly highlight the need for reliable, rapid, and cost-effective screening methods to identify edited plants regenerated from protoplasts subject to transfection with CRISPR/Cas9 Ribonucleoproteins (RNPs)(line 74-76).

Section Materials and methods:

Comments 11.  The sgRNA naming is inconsistent: (2) is repeated twice (lines 117–119).

Response 11. We agree and have corrected the manuscript accordingly.

Comments 12. Conditions for callus induction and Agrobacterium infection are only referenced, not described. At least a brief summary (e.g., media composition, selection agent concentration) should be included for reproducibility.

Response 12.  Actively proliferating Agrobacterium cultures, pre-induced with 100 μM acetosyringone (AS) for 3 hours, were re-suspended in liquid GS1CA medium (Franks et al., 1998) containing AS to an optical density at 600 nm (OD600) of 0.3–0.45. These bacterial suspensions were then combined with approximately 5 g of embryogenic callus in a total volume of 20 mL. The mixture was gently agitated at 60 rpm for 10 minutes at 25 °C, subsequently centrifuged, blotted on sterile Whatman paper, and transferred onto solid GS1CA medium. Co-cultivation was carried out in the dark at 25 °C for 48 hours. Following co-cultivation, the callus was washed with liquid GS1CA medium supplemented with 1 g/L Timentin, centrifuged, blotted dry, and transferred onto solid GS1CA medium containing 1 g/L Timentin. Cultures were maintained in the dark at 25 °C for 4 weeks. After this period, calli were subcultured monthly onto fresh GS1CA medium supplemented with 1 g/L Timentin and 150 mg/L Kanamycin, under the same dark conditions at 25 °C, for a total duration of 8 months. Mature embryos at the torpedo stage were then transferred to NN medium (Nitsch & Nitsch, 1969) supplemented with 25 mg/L Kanamycin and incubated under a 16-hour photoperiod to promote differentiation and germination. Regenerated transgenic shoots were transferred to WP medium (McCown & Lloyd, 1981) and subcultured at two-month intervals.

Franks T, Gang He D, Thomas M (1998) Regeneration of transgenic Vitis vinifera L. Sultana plants: genotypic and phenotypic analysis. Mol Breed 4:321–33325.

Nitsch JP, Nitsch C (1969) Haploid plants from pollen grains. Science 163:85–87. https:// doi. org/ 10. 1126/ science. 163. 3862. 8526.

McCown BH, Lloyd G (1981) Woody plant medium (WPM)–a mineral nutrient formulation for microculture of woody plants pecies. Hortic Sci 16:453

Comments 13. Only Nanodrop quantification is mentioned (line 147), which may not be accurate for purity. Fluorometric quantification (e.g., Qubit) would be more reliable.

Response 13. Yes, we agree. Indeed, we used a Nanodrop to assess DNA quality. Although it was not mentioned in the manuscript, we also used a fluorometer (Qubit). We have now added this information (line 152-154).

Comments 14.           Primer sequences are listed inline; better presentation would be a table of primers with expected product sizes.

Response 14. This information is provided in Table S1.

Comments 15.           The HRM assay mentions only “two technical replicates per sample” (line 171). For reliability, biological replicates should also be specified.

Response 15.  It is important to note that this method is particularly suitable for an initial screening phase, during which in vitro plantlets can be preliminarily assessed for the presence of the desired mutation (as illustrated in panels a and c of Figure 2). At this stage, generating biological replicates for each putatively edited line would be both labor-intensive and of limited utility. A second application of this method, shown in panel b of Figure 2, involves the evaluation of biological replicates from previously characterized, genome-edited lines. This approach serves to confirm their genetic identity prior to undertaking large-scale experiments, such as those conducted under greenhouse conditions. In this context, multiple biological replicates are readily available for analysis.

 

Comments 16.           The HRM ramp (line 176) is described, but more details (e.g., data acquisition frequency, number of readings per °C increment) would improve reproducibility.

Response 16. We agree that additional details about the HRM ramp are important to ensure reproducibility. We have now included the data acquisition frequency and the number of readings per °C increment in the revised manuscript (line 183-184).

Comments 17.           The description of PCA is unnecessarily mathematical (lines 204–211) for a methods section. A concise description of how PCA was applied to HRM data would suffice, with citations for statistical background.

Response 17. We have removed several sentences to improve clarity and have revised the section to make it more concise.

Comments 18.           The workflow for curve normalization (lines 184–191) should include justification for the chosen 80% confidence threshold.

Response 18. This threshold was selected based on preliminary tests, which showed that it provided a good balance between sensitivity and specificity in distinguishing subtle variations among melting curves. Additionally, it is consistent with thresholds used in similar HRM-based studies (line 199-203).

Section Results:

Comments 19. Many passages (e.g., lines 321–337, 356–361) restate figure legends in full. This makes the results section redundant and harder to follow. Figure legends should carry descriptive detail; the main text should focus on interpretation.

Response 19. Lines 321–337 (in the revised manuscript lines 332-349) refer to the legend of Figure 2, where we explain the PCA graphs and the nomenclature given to the samples based on the sgRNA used, the edited gene, and the cultivar to which the edited plants belong. We agree that it is a long legend, full of redundancy and we shorten it.

Comments 20. In 3.1, only n=2 samples are tested per genotype (lines 263–264). This is too low to establish robustness of HRM specificity. The authors should include more biological replicates or at least acknowledge this limitation.

Response 20. It is important to note that, in Section 3.1, we were working with wild-type Vitis vinifera cultivars, some of which have a published reference genome online available (https://www.grapegenomics.com/pages/all.php). Therefore, no mutations were expected at the target sites in the WT cultivars under analysis. In general, a minimum of three biological replicates is required only for quantitative molecular characterization assays, to ensure statistical reliability, such as gene expression analysis where transcript abundance is quantified and variability between biological replicates may be large.

Comments 21. For greenhouse and in vitro plants (3.2), the number of biological and technical replicates is unclear.

Response 21. The screening was carried out in three different situations. In the first, VviEPFL9-1 was assessed on a set of 19 in vitro Kober edited plants (2 technical replicates each), while in the second on 7 biological replicates of 'Sugraone' wt and edited lines cultivated in greenhouse (2 technical replicates each). In the third, the gene VviEPFL9-2 was assessed in 12 KO lines of the variety ‘Syrah’ (2 technical replicates each) and 4 of the rootstock Kober 5BB (2 technical replicates each).

Comments 22. The PCA descriptions are overly detailed in the text (lines 286–297, 303–317), while the figures themselves are complex. A better strategy is to summarize main findings in text and leave specifics to figure legends and supplements.

Response 22. The main findings are reported in the Results section, where we highlight how the cluster separation obtained through PCA is consistent with the experimental data.

Section Discussion:

Comments 23. Discuss whether Sanger/NGS was used (or should be used) to confirm HRM-based classification.

Response 23. In Conclusion section we emphasized the importance of using sequencing techniques to accurately confirm mutations (line 474-480).

 

Comments 24. Reframe HRM’s role as a preliminary screening tool that reduces the number of samples requiring costly NGS validation.

Response 24. We have reformulated this concept in the Conclusions section, highlighting HRM as a valuable preliminary screening tool that reduces the number of samples requiring costly NGS validation (line 474-480).

Comments 25. Highlight how HRM could accelerate grapevine editing pipelines.

Response 25.  High-Resolution Melting (HRM) analysis can significantly streamline the screening phase of the grapevine genome editing pipeline, particularly in the context of NGT-1 (New Genomic Techniques–1) applications, where selectable marker genes, such as those conferring antibiotic resistance, are not employed. In such cases, where large numbers of regenerated plants must be analyzed, HRM serves as a rapid, sensitive, and cost-effective primary screening tool for the identification of edited individuals.

 Section Conclusion:

Comments 26. Clarify novel contributions of this study.

Response 26. Please refers to comment 5.

 

Comments 27. State limitations more explicitly.

Response 27. We have added further details regarding the features and limitations of the HRM technique used in this study. These additions help clarify both the strengths of HRM as a rapid and cost-effective screening tool and its limitations (please refers to lines 491-498)

Comments 28. Add future research/applications.

Response 28. Thank you for your valuable comment. Future research on the application of High Resolution Melting (HRM) analysis in detecting edited grapevine could focus on enhancing sensitivity and throughput for identifying small insertions, deletions, and single nucleotide changes introduced by gene editing tools such as CRISPR/Cas9. HRM could be further optimized for high-throughput screening of edited regenerants in breeding programs, enabling rapid and cost-effective pre-screening before sequencing. Additionally, HRM may be explored for its potential to detect off-target (line 461-476).

 

Comments 29. Refine claims about PCA to avoid overstatement.

Response 29. Thank you for your helpful suggestion. We have refined the claims regarding PCA to avoid overstatement; please see the revised text in lines 500-502.

Comments 30. Link findings to grapevine breeding relevance.

Response 30. We have revised the manuscript to better emphasize the relevance of our findings for grapevine breeding (please see lines 502-504).

Comments 31. Improve clarity and conciseness of wording.

 Response 31. Please see Conclusion section.

 

 

 

 

Reviewer 2 Report

The manuscript “High-Resolution Melting (HRM) analysis for screening edited lines: a case study in Vitis spp.” described a post-PCR and fluorescence based method to distinguish the WT allele and the edited allele of CRISPR/Cas9 generated mutants in grapevine plants. This provides an alternative affordable and easy method to identify the desired edits generated by CRISPR/Cas9 technology. 

Other questions

1. Did the authors try this HRM method to distinguish the mosaic or heterogenous mutation happen in Vitis spp.? 

     2. How did the authors make sure the primer designs were optimized for amplification of VviEPFL9-1 and           VviEPFL9-2 during the HRM assays?

Minor errors

Page 3, Line 126, in vitro, please italicize

Page 3, Line 129, in vitro, please italicize

Page 3, Line 130, in vitro, please italicize

Page 12, Line 424, incorporated, wrong spelling, please correct that

Page 12, Line 452, in vitro, please italicize

Author Response

3. Point-by-point response to Comments and Suggestions for Authors

Comments 1: Did the authors try this HRM method to distinguish the mosaic or heterogenous mutation happen in Vitis spp.?    Response 1. Thank you for the question. No, we did not specifically test this HRM method on mosaic or heterogeneous mutations in Vitis spp.   Comments 2: How did the authors make sure the primer designs were optimized for amplification of VviEPFL9-1 and VviEPFL9-2 during the HRM assays? Response 2. We verified the specificity of the primers testing different genotypes of grapevine plants belonging to different Vitis spp setting –up a HRM method. For each gene the melting curves of the four genotypes were overlapping, thus proving that no polymorphism was present among the four genotypes in the amplified region, nor different alleles within the genotype. Moreover, melting peaks were single and sharp indicating that the primers chosen to amplify the selected regions were very specific. Comments 3 Page 3, Line 126, in vitro, please italicize Response 3. Done Comments 4 Page 3, Line 129, in vitro, please italicize Response 4. Done Comments 5 Page 3, Line 130, in vitro, please italicize Response 5. Done Comments 6 Page 12, Line 424, incorporated, wrong spelling, please correct that Response 6. Done Comments 7 Page 12, Line 452, in vitro, please italicize Response 7. Done

 

Author Response File: Author Response.docx

Reviewer 3 Report

The methodology presented should impact the analysis of the population of mutants.

The paper reports a method for screening targeted mutant populations using high-resolution melting in Vitis vinifera. It is well organized. However, the resolution of figures needs to be improved.

Lines 66, 69. Defining acronyms can be useful.

Line 120. Determining the Agrobacterium strain is required.

Line 122. The age of the plant used is required.

Line 127. It should be useful to add the WP medium components if they are not too large.

Lines 129-133. Defined methodologies for DNA preparation for sequencing are required.

Table S3. Double-check the label for the sequenced reads in the table. For instance, the first pair of reads.

Figure 1. Improvement of the readable of figure is suggested. An increase in the thickness of the lines may help.

It is suggested to maintain consistency between the labels of the figure panels and the citing figure within the main text. For instance, line 286 (Figure 2A) vs line 321 (Figure 2a).

Line 324. It mentions a “CRISPResso software” but it is not in the methods section.

Line 351-354. It would be useful to reflect in the methods section the process used for re-extraction and provide recommendations for high-quality DNA sample preparation.

Discussion section. It would be useful to elaborate a brief discussion on the impact of high-quality DNA on HMR analysis. It should also be valuable to emphasize the difference in approach reported in this work compared to the previously reported approach for HMR analysis of mutants.

There are typos in the manuscript, for instance,  123, 382, 425, 47,

 

Author Response

3. Point-by-point response to Comments and Suggestions for Authors

Comments 1: Lines 66, 69. Defining acronyms can be useful.

Response 1: Thank you for pointing this out. We agree with this comment. We defined the acronym NGT in line 39, abstract section, and in line 69.

Comments 2: Line 120. Determining the Agrobacterium strain is required.

Response 2: Done. The experiments were carried out using Agrobacterium tumefaciens strain EHA105 (line 122).

 

Comments 3. Line 122. The age of the plant used is required.

Response 3. The in vitro plants analyzed for screening were one month old and 5–6 cm tall. The greenhouse plants were one year old when they were analyzed.

Comments 4. Line 127. It should be useful to add the WP medium components if they are not too large.

Response 4. The WP medium is a commercial product that contains macro- and micronutrients and vitamins as described by Lloyd and McCown (1981). Here the link of the product https://phytotechlab.com/mwdownloads/download/link/id/4568

Comments 5. Lines 129-133. Defined methodologies for DNA preparation for sequencing are required.

Response 5. The procedure was described in Clemens et al 2022 as cited in the paper ). In the grapevine edited lines, a region of the gene VvEPFL9-1 containing the site targeted by the sgRNA/Cas9 complex, was amplified with primers VvEPFL9-1_fw and VvEPFL9-1_rv (see Table S1) both elongated with overhang Illumina adapters. PCR was carried out in 20 μl final volume containing 1 × PCR BIO (Resnova, Rome, Italy), 0.4 μM of each primer and 30 ng of genomic DNA. The Illumina library was sequenced on an Illumina MiSeq platform at the Sequencing Platform Facility of Fondazione Edmund Mach (San Michele all’Adige, Italy). The same pipeline is also described in Shahbaz et al., 2025 for the VviEPFL9-2 gene.

Comments 6. Table S3. Double-check the label for the sequenced reads in the table. For instance, the first pair of reads.

Response 6. Done

Comments 7. Figure 1. Improvement of the readable of figure is suggested. An increase in the thickness of the lines may help.

Response 7. Agree. Done

Comments 8. It is suggested to maintain consistency between the labels of the figure panels and the citing figure within the main text. For instance, line 286 (Figure 2A) vs line 321 (Figure 2a).

Response 8. Agree. Done, we corrected in line 286, 304,344.

Comments 9. Line 324. It mentions a “CRISPResso software” but it is not in the methods section.

Response 9. Agree. We added details about it in line 138-140.

Comments 10. Line 351-354. It would be useful to reflect in the methods section the process used for re-extraction and provide recommendations for high-quality DNA sample preparation.

Response 10. Thank you for this helpful suggestion. We agree that describing the re-extraction process and providing recommendations for obtaining high-quality DNA would improve the clarity and reproducibility of the method. Accordingly, we have added a detailed description of the re-extraction procedure in the Methods section (DNA extraction), including the use of small, healthy green in vitro leaves and optimized handling conditions. We have also included practical recommendations for high-quality DNA preparation, such as tissue selection, freshness, and fluorometric quantification.

 

Comments 11. It would be useful to elaborate a brief discussion on the impact of high-quality DNA on HMR analysis. It should also be valuable to emphasize the difference in approach reported in this work compared to the previously reported approach for HMR analysis of mutants.

Response 11. We agree that the quality of genomic DNA can significantly influence the performance of HRM analysis. In the revised manuscript, we have added a brief discussion on the impact of DNA purity and integrity on melting curve resolution and specificity (line 497-507).

Comments 12. There are typos in the manuscript, for instance, 123, 382, 425, 47,

 Response 12. Done

 

 

 

 

 

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Dear Authors,

Thank you for making significant revisions to the manuscript and improving its quality. 

 

All my comments and concerns are well addressed in the revised version.