Generation of isogenic models of Angelman syndrome and Prader-Willi syndrome in CRISPR/Cas9-engineered human embryonic stem cells

Rachel B. Gilmore; Dea Gorka; Christopher E. Stoddard; Pooja Sonawane; Justin Cotney; Stormy J. Chamberlain

doi:10.1371/journal.pone.0311565

Peer Review History

Original SubmissionOctober 2, 2023
23 Jan 2024 Decision Letter - Osman El-Maarri, Editor PONE-D-23-32075Generation of isogenic models of Angelman syndrome and Prader-Willi syndrome in CRISPR/Cas9-engineered human embryonic stem cellsPLOS ONE Dear Dr. Cotney, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. In fact several comments where pointing that better characterization and replication of experiments are needed. Please submit your revised manuscript by Mar 08 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. 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Thank you for stating the following in the Acknowledgments Section of your manuscript: "This work was supported by National Institutes of Health (NIH) grant T32HG010463 (R.B.G.), R35GM119465 (J.C.), R01 HD099975 (J.C. and S.J.C.), and R01HD094953 (S.J.C.). This study makes use of data generated by the DECIPHER community. A full list of centres who contributed to the generation of the data is available from https://deciphergenomics.org/about/stats and via email from contact@deciphergenomics.org. DECIPHER is hosted by EMBL-EBI and funding for the DECIPHER project was provided by the Wellcome Trust [grant number WT223718/Z/21/Z]. We thank Yaling Liu from the University of Connecticut Cell and Genome Engineering Core for her help with expansion and storage of stem cell lines used in this study. We thank Dr. Judy Brown and Lisa LaBelle from the Center for Genome Innovation at the University of Connecticut Institute for Systems Genomics for their help in performing CytoSNP analysis." 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Thank you for stating the following in the Competing Interests section: "S.J.C. is now an employee of F. Hoffmann-La Roche AG.". Please confirm that this does not alter your adherence to all PLOS ONE policies on sharing data and materials, by including the following statement: ""This does not alter our adherence to PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests). If there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared. Please include your updated Competing Interests statement in your cover letter; we will change the online submission form on your behalf. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Partly Reviewer #2: Yes Reviewer #3: Yes Reviewer #4: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: No Reviewer #2: Yes Reviewer #3: N/A Reviewer #4: No ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes Reviewer #4: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes Reviewer #4: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: This manuscript describes the production of new isogenic ES cell lines for modelling Angelman syndrome and Prader Willi syndrome. These ES cell lines will be very useful for the community studying these syndromes. I found the manuscript easy to read and appreciate the work that has gone into generating these lines. My main concern is that the claims made about the qRT-PCR is not well-replicated or analysed in a statistically rigorous fashion. For the most part there aren't replicates shown on the graphs, but rather just one replicate per cell line. Without replicates, here this would be samples of RNA from different times for the same cell line, statistical analyses are not possible and therefore the reader is left to interpret the qRT-PCR that has small fold changes without the benefit of knowing the underlying variation, and therefore are left to just go with the interpretation of the authors. As it stands, reductions of 50% in pluripotency factor expression is not viewed as meaningful, whereas the effect on imprinted gene expression of 50% is (because it is expected). So some further statistics and replication would be very important. I would like to see that all qRT-PCR analyses are replicated i.e. increase the replicate numbers to a minimum of 3, and then statistical analyses are performed. I am concerned that the control H9 cells may just be the parental line, rather than having gone through the clonal cell line stress similar to the edited samples. This is probably necessary considering that they see reduction in several pluripotency factors measured in Fig 2A in all edited lines. Perhaps this is a side effect of clonal line generation. I would also be interested to know the interpretation of why UBE3A-ATS is down in the mat deleted neurons. This transcript is on the paternal allele so should be retained. As a more minor point, the discussion is highly repetitive with the results. I think this could be trimmed quite a lot so that the results are discussed rather than repeated. In this case, given that the manuscript focuses on creation of new reagents, it would likely be quite a short discussion. Reviewer #2: This is a very well written manuscript on the generation of isogenic hESC lines as tools to study Angelman syndrome and Prader Willi syndrome. The work appears scientifically sound and will provide a valuable resource to the community. I have one major comment: It does not make sense to me that only one line was chosen for neuronal differentiation. Given that only four lines have been established and were characterized, all four should be subjected to all experiments, including the neuronal differentiation. I have a few minor comments: Line 43: Hypopigmentation is not a symptom of AS per se. Only in large deletion cases, involving the OCA2 gene, this is the case. Line 47: PWS is not considered a "multigenic" disorder, but rather a "contiguous gene disorder". Line 49: The potential contribution of the MAGEL2 gene (and related Schaaf-Yang syndrome) should also be included. Reviewer #3: In this study, Gilmore et al. report the generation of isogenic hESC models of Angelman syndrome and Prader-Willi syndrome using CRISPR/Cas9 technology. The authors transfected H9 ES cells with a plasmid encoding CRISPR/Cas9 and gRNA targeting GOLGA8 and screened for puromycin resistant colonies. The survival colonies were further screeded for the reduction of UBE3A expression and gene copy number at about 50% as inducers for single allelic genomic deletion at the 15q11-13 region. The parent of origin of the genomic deletion was determined by methylation analysis at the Prader-Willi Syndrome Imprinting Center on paternal chromosome. As the result, two colonies with maternal deletion and one colony with paternal deletion were identified. The approximate size of the deletion in each colony was further determined by CytoSNP analysis. The authors went on to evaluate the pluripotency of one hESC line and its potential to differentiate into neuronal cells. Overall, the authors report two isogenic hESC lines with megabase-scale deletion modeling Angelman syndrome and one for Prader-Willi syndrome. The existence of such hESC models should benefit the understanding of disease mechanisms of these disorders and future therapeutic discoveries. However, several concerns remain to be further addressed as listed below. Major comments: 1. In this study, mutant cells were screened for puromycin resistance. However, it is unclear whether the positive cell lines are stable lines with Cas9 and puromycin expression. If so, the expression of Cas9 and gRNA may cause the deletion of the additional copy of genome, thus resulting in homozygous deletion in the same region in later time. Therefor it is important to demonstrate the three characterized mutant lines are free of Cas9 and gRNA expression. 2. Results reported in Figure 3 and 4, Supplemental Figure 4, were conducted with only one or two repeats. It is important to increase n to provide more accurate measurement and report proper statistical analysis. 3. The authors evaluate the neuronal differentiation of H9Δmat15q_1 ESCs (Figure 4A, B and Supplemental Figure 4). As shown in Figure 4B, the levels of UBE3A-ATS and Pax6 in differentiated H9Δmat15q_1 cells were lower than H9 control cells, suggesting a lower potential of H9Δmat15q_1 ESCs in differentiating into neuronal cells. This possibility should be further investigated. In addition, PAX6 was the only neuronal maker evaluated. In fact, PAX6 is frequently used as a marker for neural progenitor cells, but not differentiated mature neurons. I suggest the authors to quantitatively reevaluate the differentiation of H9Δmat15q_1 ESCs using markers for mature neurons by immunofluorescence staining. Minor comments: 1. H9Δmat15q_1 and H9Δmat15q_2groups were shown in the same color. It will be helpful to distinguish them using different colors. Reviewer #4: This manuscript describes the derivation and characterization of three new hESC models of Angelman and Prader-Willi syndromes isogenic to the well characterized H9 ESC line. These are models of the large deletions and are challenging to generate by CRISPR/Cas9 approaches but the authors were successful by screening a large number of clones for possible large deletions. I have a few suggestions to improve rigor. 1. Statistical tests should be performed for the results graphed in figures 2,3 and 4. 2. Some of the experiments appear to only include 1 replicate because there are no error bars. These analyses should be repeated, with error bars and statistics performed. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No Reviewer #3: No Reviewer #4: Yes: Janine LaSalle ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0311565.r001
Revision 1
25 Jun 2024 Author Response AUTHOR RESPONSE TO REVIEWERS We thank the reviewers for their helpful comments and constructive feedback, which has aided us in strengthening the rigor and clarity of the manuscript. While reviewers recognized the utility of these lines and their importance to the field, reviewers were generally concerned with the low number of biological replicates, lack of statistical analysis, and exclusion of a subset of the cell lines generated from neuronal differentiation. As such, we have included three biological replicates for qPCR analyses, included statistical analysis of qPCR results, and provided characterization of all cell lines as mature 10-week differentiated neurons. Please find our detailed response to all reviewer comments (in blue) below. Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Partly Reviewer #2: Yes Reviewer #3: Yes Reviewer #4: Partly 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: No Reviewer #2: Yes Reviewer #3: N/A Reviewer #4: No 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes Reviewer #4: Yes 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes Reviewer #4: Yes 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: This manuscript describes the production of new isogenic ES cell lines for modelling Angelman syndrome and Prader Willi syndrome. These ES cell lines will be very useful for the community studying these syndromes. I found the manuscript easy to read and appreciate the work that has gone into generating these lines. My main concern is that the claims made about the qRT-PCR is not well-replicated or analysed in a statistically rigorous fashion. For the most part there aren't replicates shown on the graphs, but rather just one replicate per cell line. Without replicates, here this would be samples of RNA from different times for the same cell line, statistical analyses are not possible and therefore the reader is left to interpret the qRT-PCR that has small fold changes without the benefit of knowing the underlying variation, and therefore are left to just go with the interpretation of the authors. As it stands, reductions of 50% in pluripotency factor expression is not viewed as meaningful, whereas the effect on imprinted gene expression of 50% is (because it is expected). So some further statistics and replication would be very important. I would like to see that all qRT-PCR analyses are replicated i.e. increase the replicate numbers to a minimum of 3, and then statistical analyses are performed. All samples have now been analyzed in three biological replicates and two-three technical replicates. For statistical analysis, a one way ANOVA with Dunnett’s test has been performed. I am concerned that the control H9 cells may just be the parental line, rather than having gone through the clonal cell line stress similar to the edited samples. This is probably necessary considering that they see reduction in several pluripotency factors measured in Fig 2A in all edited lines. Perhaps this is a side effect of clonal line generation. We believe this comment is actually in reference to Fig 3A. After repeating the experiment with three biological replicates, we observe only one statistically significant reduction of a pluripotency gene, SOX2. This is observed in the two Angelman-like lines. Whether this is biological or an artifact of this system would require further examination. As most pluripotency genes show no significant difference between the edited lines and the parental H9 line, we don’t believe the genome editing had a drastic effect on the pluripotency status of these lines. We would anticipate further systematic characterization of these lines to support this hypothesis based on the observed morphology, behavior, and ability to differentiate into the neuronal lineage of all edited lines. I would also be interested to know the interpretation of why UBE3A-ATS is down in the mat deleted neurons. This transcript is on the paternal allele so should be retained. We do not observe a statistically significant decrease in UBE3A-ATS in maternal deletion lines after analyzing additional biological replicates and performing statistical analysis (Figure 4C and S5 Fig). As a more minor point, the discussion is highly repetitive with the results. I think this could be trimmed quite a lot so that the results are discussed rather than repeated. In this case, given that the manuscript focuses on creation of new reagents, it would likely be quite a short discussion. The discussion was trimmed to remove repetitive sections, while still focusing on points not clarified in the results section. Reviewer #2: This is a very well written manuscript on the generation of isogenic hESC lines as tools to study Angelman syndrome and Prader Willi syndrome. The work appears scientifically sound and will provide a valuable resource to the community. I have one major comment: It does not make sense to me that only one line was chosen for neuronal differentiation. Given that only four lines have been established and were characterized, all four should be subjected to all experiments, including the neuronal differentiation. All cell lines described have now been included in neuronal differentiation and characterization. We have only included one of the Angelman-like lines, H9Δmat15q11-q13_1, in the main figure (Fig 4) for a few reasons. The deletion harbored by the H9Δmat15q11-q13_1 is a more common deletion found in AS patients compared to the deletion in the H9Δmat15q11-q13_2 line. Additionally, for one of the biological replicates of the H9Δmat15q11-q13_2 line we obtained very low yield of RNA precluding reliable qPCR measurements for one biological replicate. We wanted to be sure this line was included in the characterization and publication but admit further replication is likely warranted, and thus we chose to move this cell line to the supplement (S5 Fig) for neuronal characterization. I have a few minor comments: Line 43: Hypopigmentation is not a symptom of AS per se. Only in large deletion cases, involving the OCA2 gene, this is the case. This point has been clarified. Line 47: PWS is not considered a "multigenic" disorder, but rather a "contiguous gene disorder". The wording has been changed. Line 49: The potential contribution of the MAGEL2 gene (and related Schaaf-Yang syndrome) should also be included. This point has been included. Reviewer #3: In this study, Gilmore et al. report the generation of isogenic hESC models of Angelman syndrome and Prader-Willi syndrome using CRISPR/Cas9 technology. The authors transfected H9 ES cells with a plasmid encoding CRISPR/Cas9 and gRNA targeting GOLGA8 and screened for puromycin resistant colonies. The survival colonies were further screeded for the reduction of UBE3A expression and gene copy number at about 50% as inducers for single allelic genomic deletion at the 15q11-13 region. The parent of origin of the genomic deletion was determined by methylation analysis at the Prader-Willi Syndrome Imprinting Center on paternal chromosome. As the result, two colonies with maternal deletion and one colony with paternal deletion were identified. The approximate size of the deletion in each colony was further determined by CytoSNP analysis. The authors went on to evaluate the pluripotency of one hESC line and its potential to differentiate into neuronal cells. Overall, the authors report two isogenic hESC lines with megabase-scale deletion modeling Angelman syndrome and one for Prader-Willi syndrome. The existence of such hESC models should benefit the understanding of disease mechanisms of these disorders and future therapeutic discoveries. However, several concerns remain to be further addressed as listed below. Major comments: 1. In this study, mutant cells were screened for puromycin resistance. However, it is unclear whether the positive cell lines are stable lines with Cas9 and puromycin expression. If so, the expression of Cas9 and gRNA may cause the deletion of the additional copy of genome, thus resulting in homozygous deletion in the same region in later time. Therefor it is important to demonstrate the three characterized mutant lines are free of Cas9 and gRNA expression. We apologize for the lack of clarity in describing the transfection and selection. The Cas9 cassette is not stably integrated in these lines; both Cas9 and puromycin resistance are expressed transiently. We added clarification to the methods section to better explain this process. 2. Results reported in Figure 3 and 4, Supplemental Figure 4, were conducted with only one or two repeats. It is important to increase n to provide more accurate measurement and report proper statistical analysis. Three biological replicates have been included for all lines and statistical analysis has been performed. 3. The authors evaluate the neuronal differentiation of H9Δmat15q_1 ESCs (Figure 4A, B and Supplemental Figure 4). As shown in Figure 4B, the levels of UBE3A-ATS and Pax6 in differentiated H9Δmat15q_1 cells were lower than H9 control cells, suggesting a lower potential of H9Δmat15q_1 ESCs in differentiating into neuronal cells. This possibility should be further investigated. In addition, PAX6 was the only neuronal maker evaluated. In fact, PAX6 is frequently used as a marker for neural progenitor cells, but not differentiated mature neurons. I suggest the authors to quantitatively reevaluate the differentiation of H9Δmat15q_1 ESCs using markers for mature neurons by immunofluorescence staining. We have now included data for neuronal differentiation of all lines (Fig 4). While the main figure only shows one of the AS-like lines, H9Δmat15q11-q13_1, an equivalent characterization of the other AS-like line, H9Δmat15q11-q13_2, is included in S5 Fig. In addition, we increased the number of neuronal genes measured by including DLG4, the gene which encodes PSD-95 protein, and MAP2 to our analysis. For clarity, we moved the neuronal genes, including PAX6, to their own graph. We also performed immunocytochemistry for NeuN & MAP2 proteins (S5 Fig), though believe that immunofluorescence experiments should only be used as a qualitative measure and thus have not quantified them in any way. Minor comments: 1. H9Δmat15q_1 and H9Δmat15q_2groups were shown in the same color. It will be helpful to distinguish them using different colors. We modified our color scheme so each cell line is distinguished by its own color throughout the manuscript. Reviewer #4: This manuscript describes the derivation and characterization of three new hESC models of Angelman and Prader-Willi syndromes isogenic to the well characterized H9 ESC line. These are models of the large deletions and are challenging to generate by CRISPR/Cas9 approaches but the authors were successful by screening a large number of clones for possible large deletions. I have a few suggestions to improve rigor. 1. Statistical tests should be performed for the results graphed in figures 2,3 and 4. As Fig 2 describes the screening process, it would be inappropriate to perform statistical analysis. The results of the screen were followed by more rigorous characterization. Additional biological replicates were added, and statistical analysis was performed on data presented in Figs 3 and 4. 2. Some of the experiments appear to only include 1 replicate because there are no error bars. These analyses should be repeated, with error bars and statistics performed. Additional biological replicates were added, error bars have been included on relevant graphs, and statistical analysis has been performed on data presented in Fig 3, Fig 4, S4, S5, and S6. 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No Reviewer #3: No Reviewer #4: Yes: Janine LaSalle [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf Manuscript and file formatting has been updated accordingly. 2. Did you know that depositing data in a repository is associated with up to a 25% citation advantage (https://doi.org/10.1371/journal.pone.0230416)? If you’ve not already done so, consider depositing your raw data in a repository to ensure your work is read, appreciated and cited by the largest possible audience. You’ll also earn an Accessible Data icon on your published paper if you deposit your data in any participating repository (https://plos.org/open-science/open-data/#accessible-data). Cell lines are available through the UConn Cell and Genome Engineering Core upon request and completion of MTA. Raw data from qPCR and analysis is provided in S2 Table. Code used for generation of graphs is publicly available on Github, as listed in the Methods section. 3. Thank you for stating the following in the Acknowledgments Section of your manuscript: "This work was supported by National Institutes Attachments Attachment Submitted filename: ResponsetoReviewers.docx https://doi.org/10.1371/journal.pone.0311565.r002
23 Jul 2024 Decision Letter - Osman El-Maarri, Editor PONE-D-23-32075R1Generation of isogenic models of Angelman syndrome and Prader-Willi syndrome in CRISPR/Cas9-engineered human embryonic stem cellsPLOS ONE Dear Dr. Cotney, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Before we move to final acceptance you need as authors to comments and satisfy one of the reviwers who asked for minor revision. Please submit your revised manuscript by Sep 06 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols. We look forward to receiving your revised manuscript. Kind regards, Osman El-Maarri, Ph.D Academic Editor PLOS ONE Journal Requirements: Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: All comments have been addressed Reviewer #2: All comments have been addressed Reviewer #3: (No Response) ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: (No Response) Reviewer #2: Yes Reviewer #3: No ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: (No Response) Reviewer #2: Yes Reviewer #3: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: (No Response) Reviewer #2: Yes Reviewer #3: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: (No Response) Reviewer #2: Yes Reviewer #3: Yes ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: (No Response) Reviewer #2: The authors have done a good job addressing the reviewers' comments. I do not have any further comments. Reviewer #3: The authors did not adequately address our only main concern. One (easy) experiment is necessary: The cell lines the authors made are likely to be an important resource for the Angelman syndrome research community. Some in the field may seek to use these cell lines to test genome editor-based therapies. The authors used plasmids encoding Cas9, the guide RNAs, and puromycin resistance. If Cas9 and/or guide RNAs are stably integrated into the genome of these cells, this has the potential to confound future experiments that use the same Cas variant and/or different guide RNAs and could lead to instability of the lines themselves. Circular plasmids, when transiently transfected, can stably integrate into the mammalian genome. A recent study nicely quantifies this phenomenon: Lim et al (https://pubmed.ncbi.nlm.nih.gov/37100816/). The authors attempted to address our main concern by writing the following in the methods section: “As the Cas9 cassette was not stably integrated into these cell lines, selection was continued for 48 hours total to select cells transiently expressing the vector containing the gRNA, Cas9 protein, and puromycin resistance” However, they provide no data nor performed any experiments to confirm that the Cas9 cassette was not stably integrated. The authors already have genomic DNA and RNA isolated from these cells. We are thus requesting that the authors perform PCR (with genomic DNA) and RT-PCR (with RNA) to confirm that Cas9, the guide RNAs, and puromycin sequences are not present in the genome or expressed in the cell lines. ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No Reviewer #3: No ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0311565.r003
Revision 2
6 Sep 2024 Author Response Only changes or additions requested by the editor were made in this final revision. https://doi.org/10.1371/journal.pone.0311565.r004
23 Sep 2024 Decision Letter - Osman El-Maarri, Editor Generation of isogenic models of Angelman syndrome and Prader-Willi syndrome in CRISPR/Cas9-engineered human embryonic stem cells PONE-D-23-32075R2 Dear Dr. Cotney, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Osman El-Maarri, Ph.D Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #3: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #3: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #3: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #3: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #3: Yes ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #3: The remaining minor concern has been adequately addressed. ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #3: No ******** https://doi.org/10.1371/journal.pone.0311565.r005
Formally Accepted
25 Oct 2024 Acceptance Letter - Osman El-Maarri, Editor PONE-D-23-32075R2 PLOS ONE Dear Dr. Cotney, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps. Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. If we can help with anything else, please email us at customercare@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Priv.-Doz. Dr. Osman El-Maarri Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0311565.r006

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