Genome-wide CRISPR interference screen identifies long non-coding RNA loci required for differentiation and pluripotency

Jeffrey R. Haswell; Kaia Mattioli; Chiara Gerhardinger; Philipp G. Maass; Daniel J. Foster; Paola Peinado; Xiaofeng Wang; Pedro P. Medina; John L. Rinn; Frank J. Slack

doi:10.1371/journal.pone.0252848

Peer Review History

Original SubmissionMay 30, 2021
9 Jul 2021 Decision Letter - Romi Gupta, Editor PONE-D-21-17843 Genome-wide CRISPR interference screen identifies long non-coding RNA loci required for differentiation and pluripotency PLOS ONE Dear Dr. Slack, 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. Please submit your revised manuscript by July 28. 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. 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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: Yes Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 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. 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(Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The authors have performed well designed and well controlled experiments in the manuscript. I do not have any suggestions for major experiments for the manuscript. Two minor suggestions: 1. Hit lncRNA and non hit lncRNA do they have any specific chromatin signature, as authors suggest a major portion of hit lncRNA acts through DNA elements. 2. FOXD3 coding gene promoter do show any change at local chromatin structure in the shRNA KD? Reviewer #2: In this study “Genome-wide CRISPR interference screen identifies long non-coding RNA loci required for differentiation and pluripotency” author developed CRISPRi and CRISPRa hESC lines and demonstrated robust repression and activation, respectively, of lncRNAs as well as protein-coding genes that effectively modulate gene expression that involved hESC lines. Author also developed an unsupervised learning approach to predict the potential mechanisms of action of lncRNA loci based on select genomic features. The current study successfully established an approach that can be applied to multiple lineage specific differentiation processes. Minor comments. 1. Figure.1 B. the represented western blots are saturated, please replace with another exposure. 2. Figure 1. author encouraged to show the expression of differentiation markers as primary evidence of the model systems. 3. Figure 7C. Author encouraged to show the knockdown FOXD3-AS1 efficiency by western blotting. 4. Author encouraged to study one more lncRNA from Figure 7A. ****** 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: Yes:** Suresh Chava [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.0252848.r001
Revision 1
16 Aug 2021 Author Response Full response to reviewers is attached in the manuscript data as it contains a plot, but is copy-pasted here for convenience. Reviewer #1: The authors have performed well designed and well controlled experiments in the manuscript. I do not have any suggestions for major experiments for the manuscript. Two minor suggestions: 1. Hit lncRNA and non hit lncRNA do they have any specific chromatin signature, as authors suggest a major portion of hit lncRNA acts through DNA elements. We thank the reviewer for this suggestion. While we did look to see if our hit lncRNA loci were closer to nearby endoderm enhancers than non-hit loci (Figure S6K), we did not initially look at specific chromatin marks. We have therefore downloaded the ChIP-seq peaks for the 4 chromatin marks studied in definitive endoderm in Loh et al. 2014—H3K27ac, H3K27me3, H3K4me3, and H3K4me2—and determined their locations in relation to our hit lncRNA loci vs. non-hit lncRNA loci. We found that hit lncRNAs were, on average, closer than non-hit lncRNA loci to definitive endoderm H3K4me3 peaks, H3K4me2 peaks, and H3K27ac peaks, though all had only moderate Mann Whitney p-values (minimum 0.073, maximum 0.136). Thus, both promoter marks (H3K4me3 and H3K4me2) and enhancer marks (H3K27ac) are somewhat enriched near hit lncRNA loci, consistent with our conclusion that many of the lncRNA loci may act as DNA elements. The only ChIP-seq profile which did not show any differences between hits and non-hits is H3K27me3, which is expected, as this mark delineates silenced (not actively expressed) regions. We have added the following plots to Figure S6: And have updated the Methods section to describe the above analysis: “We also determined the distance from the TSS to the closest endoderm-specific enhancer as defined by Loh et al., 2014 (their Table S5) as well as the distance from the TSS to the closest definitive endoderm H3K27ac, H3K27me3, H3K4me2, and H3K4me3 peaks (peak calls downloaded from the Cistrome database) (Liu et al., 2011).” 2. FOXD3 coding gene promoter do show any change at local chromatin structure in the shRNA KD? While we did not examine the local chromatin structure of the FOXD3 coding gene promoter upon FOXD3-AS1 shRNA KD, our group is currently exploring the mechanism behind FOXD3-AS1 regulation of pluripotency, including examining changes in chromatin landscape upon FOXD3-AS1 KD. Given that similar phenotypes were observed in both DNA- (CRISPRi) and RNA-based (shRNA) methods of FOXD3-AS1 KD, we believe that FOXD3-AS1 acts as a functional lncRNA as opposed to a DNA element. This hypothesis is supported by the recent study by Guo et al. (2020; cited in our manuscript) that characterized FOXD3-AS1 as acting in the cytoplasm to block the interaction between �-TrCP and phosphorylated �-catenin, leading to suppressed �-catenin degradation and WNT activation. We are currently performing ChIP-seq experiments to examine potential chromatin changes mediated at the FOXD3/FOXD3-AS1 promoter locus and are investigating the role that FOXD3 may play in FOXD3-AS1-mediated control of pluripotency. Reviewer #2: In this study “Genome-wide CRISPR interference screen identifies long non-coding RNA loci required for differentiation and pluripotency” author developed CRISPRi and CRISPRa hESC lines and demonstrated robust repression and activation, respectively, of lncRNAs as well as protein-coding genes that effectively modulate gene expression that involved hESC lines. Author also developed an unsupervised learning approach to predict the potential mechanisms of action of lncRNA loci based on select genomic features. The current study successfully established an approach that can be applied to multiple lineage specific differentiation processes. Minor comments. 1. Figure.1 B. the represented western blots are saturated, please replace with another exposure. We thank the reviewer for this suggestion. Unfortunately there are no additional Western blot exposures that were taken, so we are unable to provide those images without performing additional experiments. All authors on this manuscript are no longer in the corresponding authors’ labs, making it difficult to repeat the experiments in a timely manner. However, we are confident that the conclusions from Figure 1B are supported by several additional data points in the paper, including Figure 1D demonstrating maintenance of GFP expression throughout differentiation in all three lineages and Figures 1E-1G demonstrating robust knockdown of both protein-coding (1E) and lncRNA (1F-G) genes. If dCas9-KRAB expression was not maintained over the course of endoderm and mesoderm differentiation, the levels of target gene repression would likely not be as strong. 2. Figure 1. author encouraged to show the expression of differentiation markers as primary evidence of the model systems. We thank the reviewer for this suggestion. The levels of pluripotency and differentiation markers observed in our engineered lines (both protein-coding and non-coding genes) were similar to those observed in wild-type H1 hESCs, as evidenced by high RT-qPCR expression of lncRNAs in the absence of targeted sgRNAs (Figure 1F-1G), as well as strong protein expression observed by FACS during the screens (Figure S4) and in individual validation experiments (Figure 4). 3. Figure 7C. Author encouraged to show the knockdown FOXD3-AS1 efficiency by western blotting. We thank the reviewer for the suggestion. Since FOXD3-AS1 does not code for a protein, we are unable to demonstrate knockdown efficiency via Western blotting, and instead showed RT-qPCR to demonstrate efficient knockdown of the RNA transcript. 4. Author encouraged to study one more lncRNA from Figure 7A. We thank the reviewer for the suggestion. The main purpose of this report is to serve as a resource for the community rather than a deep dive into specific lncRNAs. We are currently investigating several lncRNAs identified from the screen (in addition to FOXD3-AS1) for their role in mediating pluripotency and differentiation. We look forward to sharing those findings in a future publication. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0252848.r002
27 Aug 2021 Decision Letter - Romi Gupta, Editor Genome-wide CRISPR interference screen identifies long non-coding RNA loci required for differentiation and pluripotency PONE-D-21-17843R1 Dear Dr. Slack, 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 for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, 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, Romi Gupta Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0252848.r003
Formally Accepted
25 Oct 2021 Acceptance Letter - Romi Gupta, Editor PONE-D-21-17843R1 Genome-wide CRISPR interference screen identifies long non-coding RNA loci required for differentiation and pluripotency Dear Dr. Slack: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. 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 plosone@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 Dr. Romi Gupta Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0252848.r004

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