Duchenne muscular dystrophy cell culture models created by CRISPR/Cas9 gene editing and their application in drug screening
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Abstract
Gene editing methods are an attractive therapeutic option for Duchenne muscular dystrophy, and they have an immediate application in the generation of research models. To generate myoblast cultures that could be useful in in vitro drug screening, we have optimised a CRISPR/Cas9 gene edition protocol. We have successfully used it in wild type immortalised myoblasts to delete exon 52 of the dystrophin gene, modelling a common Duchenne muscular dystrophy mutation; and in patient’s immortalised cultures we have deleted an inhibitory microRNA target region of the utrophin UTR, leading to utrophin upregulation. We have characterised these cultures by demonstrating, respectively, inhibition of dystrophin expression and overexpression of utrophin, and evaluating the expression of myogenic factors (Myf5 and MyH3) and components of the dystrophin associated glycoprotein complex (α-sarcoglycan and β-dystroglycan). To demonstrate their use in the assessment of DMD treatments, we have performed exon skipping on the DMDΔ52-Model and have used the unedited DMD cultures/ DMD-UTRN-Model combo to assess utrophin overexpression after drug treatment. While the practical use of DMDΔ52-Model is limited to the validation to our gene editing protocol, DMD-UTRN-Model presents a possible therapeutic gene edition target as well as a useful positive control in the screening of utrophin overexpression drugs.
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##Author Response
###Reviewer #1:
Summary:
In this paper, the authors utilize CRISPR-Cas9 to generate two different DMD cell lines. The first is a DMD human myoblast cell line that lacks exon 52 within the dystrophin gene. The second is a DMD patient cell line that is missing miRNA binding sites within the regulatory regions of the utrophin gene, resulting in increased utrophin expression. Then, the authors proceeded to test antisense oligonucleotides and utrophin up-regulators in these cell lines.
Overall opinion (expanded in more detail below).
The paper suffers from the following weaknesses:
- The protocol used to generate the myoblast cell lines is rather inefficient and is not new.
- Many of the data figures are of low quality and are missing proper controls (detailed in points 5,7,10, 12, 13,14)
Detailed critiques:
- The…
##Author Response
###Reviewer #1:
Summary:
In this paper, the authors utilize CRISPR-Cas9 to generate two different DMD cell lines. The first is a DMD human myoblast cell line that lacks exon 52 within the dystrophin gene. The second is a DMD patient cell line that is missing miRNA binding sites within the regulatory regions of the utrophin gene, resulting in increased utrophin expression. Then, the authors proceeded to test antisense oligonucleotides and utrophin up-regulators in these cell lines.
Overall opinion (expanded in more detail below).
The paper suffers from the following weaknesses:
- The protocol used to generate the myoblast cell lines is rather inefficient and is not new.
- Many of the data figures are of low quality and are missing proper controls (detailed in points 5,7,10, 12, 13,14)
Detailed critiques:
- The title needs to be changed. The method used by the authors is inefficient. The title should instead focus on the two cell lines generated.
We appreciate the reviewer’s comments: thanks to them, we have realized the focus of the manuscript should be in the new models we described and less in the methodology used to create them.
Originally, we wanted to share the problems we faced when applying new CRISPR/Cas9 edition techniques to myoblasts: our conversations with other researchers in the field confirmed that many were having similar problems. However, the reviewer is right in the fact that there are many ways around this problem. We do describe ours and we are working in a new version of the manuscript with additional data to characterize our new models further and where the method used to create them, although included, is not the main focus of the manuscript. In this new version we will change the title accordingly.
- Line 104: The authors declare that the efficiency of CRISPR/Cas9 is currently too low to provide therapeutic benefit for DMD in vivo. There are lots of papers that show efficient recovery of dystrophin in small and large animals following CRISPR/Cas9 therapy. The authors should cite them properly.
Thank you for your appreciation. We have reviewed the literature again to include new evidences of efficient dystrophin recovery as well as other studies with lower efficiency.
- Figures 1, 2,3, and 4 can be merged into one figure.
- Figure 2A and 2B can be moved to supplementary.
- Figure 2C and 2D are not clear. Are the duplicates the same? Please invert the black and white colors of the blots.
Thank you for your comments. We have inverted the colors of the blots and changed the marks used in figure 2C and 2D to clarify that duplicates are indeed the same sample, assayed in duplicates. We have also merged figures 1 and 4 and moved figures 2 and 3 to supplementary in this new version.
- Figure 3: In order to optimize the efficiency of myoblast transfection, the plasmids containing the Cas9 and the sgRNA should have different fluorophores (GFP and mCherry). This approach would increase the percentage of positive edited clones among the clones sorted.
We think the reviewer may have misunderstood our methodology: we are not using a plasmid with the Cas9 and another with the sgRNA, we are using two plasmids, both containing Cas9 and each a different sgRNA. We did try to use two different plasmids, one expressing GFP and one expressing puromycin resistance, but we found out that single GFP positive cell selection plus puromycin selection was too inefficient. We could have tried with two different fluorophores, but we tested the tools we had in our hands first and were successful at obtaining enough clones to continue with their characterization, so we did so instead of a further optimization to our editing protocol.
- Figure 4A: In the text, the authors state that only 1 clone had the correct genomic edit, but from the PCR genotyping in this figure shows at least 2 positive clones (number 4 and 7).
Thank you for your appreciation. As you said, we got two positive clones (as we also indicate in figure 3B) but we completed the full characterization of one of them (clone number 7= DMD-UTRN-Model). In the new version of the manuscript we explain this further.
- Figure 4C: The authors should address whether one or both copies of the UTRN gene was edited in their clones.
Thank you for your comment. Both copies of the UTRN gene were edited in our clones. We have included this information both in the text and in the figure 4 legend.
- Figure 4 B and D: The authors should report the sequence below the electropherograms.
Thank you for this correction, we have included the sequence under the electropherograms.
- Figure 5B: This western blot is of poor quality. Also, the authors should specify that the samples are differentiated myoblasts. Lastly, a standard protein should be included as a loading control.
Thank you for your comment. Poor quality of dystrophin and utrophin western blots was the main reason to validate a new method in our laboratory to measure these proteins directly in cell culture (1) like an alternative to western blotting. Since then, the myoblot method has been routinely used by us and in collaboration with other groups and companies. We included the western blot as it is sometimes easier for those used to this technique to be able to assess a blot in which there is no dystrophin expression. As you pointed out, our samples were all differentiated myotubes, not myoblasts, and we have modified this accordingly. Thank you very much for pointing out this mistake
On the other hand, as described in the methods, Revert TM 700 Total Protein Stain (Li-Cor) and alpha-actinin were included as standards in dystrophin and utrophin western blots, respectively.
- Figure 5E: We would like to see triplicates for the level of Utrophin expression.
We thank the reviewer for his/her recommendation, but we do not consider western blotting a good quantitative technique, we have included western blots to show the expression/absence of protein at the same level. We have included many more replicates than needed to show at the level of utrophin by myoblots. We acknowledge that western blotting is the preferred method for some reviewers, so in the new version of our manuscript we clearly indicate the value we give to each technique, being myoblots our choice for quantification.
- Figure 6: A dystrophin western blot should be included to demonstrate protein recovery following antisense oligonucleotide treatment. Also, the RT-PCR data could be biased as you can have preferential amplification of shorter fragments.
Thank you for your recommendation but as we have explained before, myoblots have been validated in our laboratory to replace western blot for accurate dystrophin quantification in cell culture.
- Figure 6A: Invert the black and white colors. The authors should also report the control sequences and sequences of the clones under the electropherograms.
Thank you for your suggestion, we have inverted the colors and added the sequences under the electropherograms.
- Figure 6B: Control myoblasts should be included in figure 5C.
Thank you for this correction, we will include control myoblasts in the new manuscript version.
- Figure S2A: Invert the black and white colors.
Thank you for your suggestion, we have inverted the colors.
Reviewer #2:
The work from Soblechero-Martín et al reports the generation of a human DMD line deleted for exon 52 using CRISPR technology. In addition, the authors introduced a second mutation that leads to upregulation of utrophin, a protein similar to dystrophin, which has been considered as a therapeutic surrogate. The authors provide a careful description of the methodology used to generate the new cell line and have conducted meticulous evaluations to test the validity of the reagents.
However, if the main purpose of this cell line is to perform drug or small molecule compound screenings, a single line might not be sufficient to draw robust conclusions. The generation of additional DMD lines in different genetic backgrounds using the reagents developed in this study will strengthen the work and will be of interest to the DMD field.
Thank you for your appreciation. We think that a well characterized immortalized culture, like the one we describe is sufficient for compound screening, as described in other recently published studies (2), (3). About the other suggestion, we have indeed used our method to generate other cultures for collaborators, but they will be reported in their own publications, as they are interested in them as tools in their own research projects.
Further, the future use of the edited DMD line with upregulated utrophin is unclear. The utrophin upregulation adds a complexity to this line that might complicate the assessment of screened compounds. In contrast, this line could be used to test if overexpression of utrophin generates myotubes that produce increased force compared to the control DMD line.
We think we may have not explained our screening platform well enough. Our suggestion is to offer our newly generated culture ALONGSIDE the original unedited culture: the original is treated with potential drug candidates, while the new one may or may not be treated, if these drug candidates are thought to act by activating the edited region (see an example in the figure below). In this case, the new culture will be a reliable positive control to the effects that may be reported in the unedited cultures by the drug candidates. We will make this clear in the new version of the manuscript.
Created with BioRender.com
In summary, while there is support and enthusiasm for the techniques and methodological approach of the study, the future use of this single line might be dubious and could be strengthened if additional lines are generated.
We share the reviewer’s enthusiasm for this approach, and we have included in the new version of the manuscript further characterization of this new cell culture that we think would demonstrate its usefulness better.
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###Reviewer #2:
The work from Soblechero-Martín et al reports the generation of a human DMD line deleted for exon 52 using CRISPR technology. In addition, the authors introduced a second mutation that leads to upregulation of utrophin, a protein similar to dystrophin, which has been considered as a therapeutic surrogate. The authors provide a careful description of the methodology used to generate the new cell line and have conducted meticulous evaluations to test the validity of the reagents.
However, if the main purpose of this cell line is to perform drug or small molecule compound screenings, a single line might not be sufficient to draw robust conclusions. The generation of additional DMD lines in different genetic backgrounds using the reagents developed in this study will strengthen the work and will be of interest to the DMD …
###Reviewer #2:
The work from Soblechero-Martín et al reports the generation of a human DMD line deleted for exon 52 using CRISPR technology. In addition, the authors introduced a second mutation that leads to upregulation of utrophin, a protein similar to dystrophin, which has been considered as a therapeutic surrogate. The authors provide a careful description of the methodology used to generate the new cell line and have conducted meticulous evaluations to test the validity of the reagents.
However, if the main purpose of this cell line is to perform drug or small molecule compound screenings, a single line might not be sufficient to draw robust conclusions. The generation of additional DMD lines in different genetic backgrounds using the reagents developed in this study will strengthen the work and will be of interest to the DMD field.
Further, the future use of the edited DMD line with upregulated utrophin is unclear. The utrophin upregulation adds a complexity to this line that might complicate the assessment of screened compounds. In contrast, this line could be used to test if overexpression of utrophin generates myotubes that produce increased force compared to the control DMD line.
In summary, while there is support and enthusiasm for the techniques and methodological approach of the study, the future use of this single line might be dubious and could be strengthened if additional lines are generated.
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###Reviewer #1:
Summary:
In this paper, the authors utilize CRISPR-Cas9 to generate two different DMD cell lines. The first is a DMD human myoblast cell line that lacks exon 52 within the dystrophin gene. The second is a DMD patient cell line that is missing miRNA binding sites within the regulatory regions of the utrophin gene, resulting in increased utrophin expression. Then, the authors proceeded to test antisense oligonucleotides and utrophin up-regulators in these cell lines.
Overall opinion (expanded in more detail below).
The paper suffers from the following weaknesses:
The protocol used to generate the myoblast cell lines is rather inefficient and is not new.
Many of the data figures are of low quality and are missing proper controls (detailed in points 5,7,10, 12, 13,14)
Detailed critiques:
The title needs to be changed. The …
###Reviewer #1:
Summary:
In this paper, the authors utilize CRISPR-Cas9 to generate two different DMD cell lines. The first is a DMD human myoblast cell line that lacks exon 52 within the dystrophin gene. The second is a DMD patient cell line that is missing miRNA binding sites within the regulatory regions of the utrophin gene, resulting in increased utrophin expression. Then, the authors proceeded to test antisense oligonucleotides and utrophin up-regulators in these cell lines.
Overall opinion (expanded in more detail below).
The paper suffers from the following weaknesses:
The protocol used to generate the myoblast cell lines is rather inefficient and is not new.
Many of the data figures are of low quality and are missing proper controls (detailed in points 5,7,10, 12, 13,14)
Detailed critiques:
The title needs to be changed. The method used by the authors is inefficient. The title should instead focus on the two cell lines generated.\
Line 104: The authors declare that the efficiency of CRISPR/Cas9 is currently too low to provide therapeutic benefit for DMD in vivo. There are lots of papers that show efficient recovery of dystrophin in small and large animals following CRISPR/Cas9 therapy. The authors should cite them properly.
Figures 1, 2,3, and 4 can be merged into one figure.
Figure 2A and 2B can be moved to supplementary.
Figure 2C and 2D are not clear. Are the duplicates the same? Please invert the black and white colors of the blots.
Figure 3: In order to optimize the efficiency of myoblast transfection, the plasmids containing the Cas9 and the sgRNA should have different fluorophores (GFP and mCherry). This approach would increase the percentage of positive edited clones among the clones sorted.
Figure 4A: In the text, the authors state that only 1 clone had the correct genomic edit, but from the PCR genotyping in this figure shows at least 2 positive clones (number 4 and 7).
Figure 4C: The authors should address whether one or both copies of the UTRN gene was edited in their clones.
Figure 4 B and D: The authors should report the sequence below the electropherograms.
Figure 5B: This western blot is of poor quality. Also, the authors should specify that the samples are differentiated myoblasts. Lastly, a standard protein should be included as a loading control.
Figure 5E: We would like to see triplicates for the level of Utrophin expression.
Figure 6: A dystrophin western blot should be included to demonstrate protein recovery following antisense oligonucleotide treatment. Also, the RT-PCR data could be biased as you can have preferential amplification of shorter fragments.
Figure 6A: Invert the black and white colors. The authors should also report the control sequences and sequences of the clones under the electropherograms.
Figure 6B: Control myoblasts should be included in figure 5C.
Figure S2A: Invert the black and white colors.
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##Preprint Review
This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 2 of the manuscript. Lee Rubin (Harvard University) served as the Reviewing Editor.
###Summary:
While the paper by Soblechero-Martín et al., may present an ultimately useful method for modifying genes in skeletal muscle, the reviewers felt that, at the current time, the robustness of the methods and the amount of data presented were insufficient. The reviews below point towards additional experiments that could be done to improve this paper.
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