Optimizing Embryo Collection for Application of CRISPR/Cas9 System and Generation of Fukutin Knockout Rat Using This Method

Round 1

Reviewer 1 Report

The article by Dong Won Seol and colleagues addresses an important issue in translational biomedicine, namely the establishment of an animal model of FCMD. This work is undoubtedly of great interest for applied science. However, while reading, a number of questions appeared.

Major

1. Has there been any research into off-target effect? It is common drawback of genome editing systems and it should be checked.

2. Following paragraph 2-5 of Materials and Methods, I conclude that there were a total of 12 rats in the experiment, which were injected with 30 embryos.  The result was that only two rats were confirmed to have a mutation in the target gene. Is this due to the fact that fukutin is essential for embryonic development, and its absence causes the death of the embryos? Are the resulting mutations passed on to subsequent generations?

3. The relationship between phenotype and genotype in founders 1 and 3 should be explained in more detail.  How consistent are the phenotypic manifestations with those described for FCMD?

4. Immunochemical detection of fukutin in muscle sections is highly recommended. Healthy and KO rats should be compared. Quantitative Western blotting can be alternative to this analysis.

Minor.

Authors should check the spelling of biological terms.

There is an extra point in line 156, and in lines 197 and 238 spaces are missing. 

The information on lines 242-243 is text from template and must be deleted.  

Author Response

We are very grateful to the reviewer for his/her valuable review. We revised the manuscript to a more complete level through valuable comments from reviewers. The modified text was written in red.

Reviewer 1

Major 1.

1. Has there been any research into off-target effect? It is common drawback of genome editing systems and it should be checked.

Thank you for your detailed comment. To confirm the off-target effect, the DNA base sequence of the target region was confirmed using the Cas-offinder program of the CRISPR RGEN tool.

There were no identical base sequences in the non-target rat genome, and as a result of expanding the mismatch to 2bp, no identical DNA sequences were identified, so it was expected that there would be no off-target effect in the transgenic offspring. Additionally, we added table 2 and marked the revised text in lines 97-98 and line 199 in red.

2. Following paragraph 2-5 of Materials and Methods, I conclude that there were a total of 12 rats in the experiment, which were injected with 30 embryos. The result was that only two rats were confirmed to have a mutation in the target gene. Is this due to the fact that fukutin is essential for embryonic development, and its absence causes the death of the embryos? Are the resulting mutations passed on to subsequent generations?

Kurahashi et al., (2005) reported fukutin deficiency in mice is lethal at embryonic day 10.5 and is essential for embryonic development [1]. As a result of our study, compared the size and genotype of fetus implanted at embryonic day 13.5 by crossing fukutin heterogeous rats, it was confirmed that the size of fukutin KO fetus was smaller than that of other genotypes. The produced founder 1 died early (at 8 weeks), so transmission of the mutation to thte next generation was not observed. The additional experiments and results we performed were added to sFig2 and written in red text on lines 318-324.

3. The relationship between phenotype and genotype in founders 1 and 3 should be explained in more detail. How consistent are the phenotypic manifestations with those described for FCMD?

The phenotype of founder #3 was not different to WT, as verified through body weight. Subsequently, Founder #3 was bred with WT to generate heterozygous offspring, whose genotypes were confirmed to be Δ5 and Δ16. Following sibling mating, no embryos with the homozygouse genotype were detected beyond embryonic day 13.5.

4. Immunochemical detection of fukutin in muscle sections is highly recommended. Healthy and KO rats should be compared. Quantitative Western blotting can be alternative to this analysis.

Due to the absence of remaining samples of founder #1, conducting further experimentx poses a challenge.

Minor.

Authors should check the spelling of biological terms.

We re-performed spell-checking of biological terms throughout, based on reviewer suggestions.

There is an extra point in line 156, and in lines 197 and 238 spaces are missing.

We revised the extra points, based on reviewer comments

.

The information on lines 242-243 is text from template and must be deleted. 

We deleted the text of template.

Reviewer 2 Report

The authors have claimed to develop a rat model for Fukutin disease. the authors have used the Crispr cas9 system to develop the knock out. The study is interesting and important for the society. The study is mostly preliminary. However, there are several demerits in the study which needs to be addressed before its final acceptance for publication. My comments are provided below

1. The authors didnot provide any evidence of the efficiency and off target effect of the Crispr cas9 gRNA.

2. The authors didnot provide any data on the level of dystroglycan?

3. The Dystroglycan complex markers should be shown in details.

4. The behavioral tests should be performed to show the effect of the knock out of DG.

5. The authors didnot show the any data in the protein level.

Moderate spelling check is needed.

Author Response

We are very grateful to the reviewer for his/her valuable review. We revised the manuscript to a more complete level through valuable comments from reviewers. The modified text was written in red.

Reviewer 2

1. The authors didnot provide any evidence of the efficiency and off target effect of the Crispr cas9 gRNA.

Thank you for your detailed comment. To confirm the off-target effect, the DNA base sequence of the target region was confirmed using the Cas-offinder program of the CRISPR RGEN tool.

There were no identical base sequences in the non-target rat genome, and as a result of expanding the mismatch to 2bp, no identical DNA sequences were identified, so it was expected that there would be no off-target effect in the transgenic offspring. Additionally, we added table 2 and marked the revised text in lines 97-98 and line 199 in red.

2. The authors did not provide any data on the level of dystroglycan?

Founder #1 died at 8 weeks old. We tried to the expression of α, β-dystroglycan; however, it was not observed due to problems with postmortem specimen.

3. The Dystroglycan complex markers should be shown in details.

Founder #1 died at 8 weeks old. We tried to the expression of α, β-dystroglycan; however, it was not observed due to problems with postmortem specimen.

4. The behavioral tests should be performed to show the effect of the knock out of DG.

Due to the absence of remaining samples of founder #1, conducting further experiments poses a challenge.

5. The authors didnot show the any data in the protein level.

Due to the absence of remaining samples of founder #1, conducting further experimentx poses a challenge.

Round 2

Reviewer 1 Report

The authors successfully answered the questions.

Author Response

Our study aimed to investigate two primary objectives.

Firstly, we aimed to determine the optimal hormone dosage for superovulation in rats and assess culture medium conditions for in vitro culture, facilitating the utilization of gene editing via CRISPR/Cas9.

Secondly, building upon these findings, we conducted experiments to produce rats with fukutin knockout.

However, only one founder exhibiting the desired phenotype was obtained. Subsequent sibling mating of the produced heterozygotes revealed embryonic lethality in the case of homozygotes.

Therefore, we are proceeding to generate muscle creatine kinase (MCK)-Cre rats and myogenic factor 5(Myf5)-Cre rats for fukutin conditional knockout.

Soon we expect to have MCK-fukutin-cKO rats (dystrophic glycolysis model) and Myf5-fukutin-cKO rats.

If the transgenic rats mentioned above are produced, we will prepare to evaluate Fukutin's function and plan to report the results in a follow-up study in the CIMB journal.

We request that you accept our paper for publication.

Reviewer 2 Report

The authors were unable to perform additional experiment due to lack of samples. The study seems preliminary without these necessary experiment. The rat model is claimed for fukutin related muscular dystrophy but the authors didnot have the data for any of the target genes. It needs extensive validation of the scientific claim about the phenotype which should be replicable.

Minor spelling mistakes are detected in the text.

Author Response

We greatly appreciate Reviewer 2's second review. We assert our thoughts on Reviewer 2's concerns. Reviewer 2

Reviewer 2

The authors were unable to perform additional experiment due to lack of samples. The study seems preliminary without these necessary experiment.

The rat model is claimed for fukutin related muscular dystrophy but the authors didnot have the data for any of the target genes.

It needs extensive validation of the scientific claim about the phenotype which should be replicable.

Response

Our study aimed to investigate two primary objectives.

Firstly, we aimed to determine the optimal hormone dosage for superovulation in rats and assess culture medium conditions for in vitro culture, facilitating the utilization of gene editing via CRISPR/Cas9.

Secondly, building upon these findings, we conducted experiments to produce rats with fukutin knockout.

However, only one founder exhibiting the desired phenotype was obtained. Subsequent sibling mating of the produced heterozygotes revealed embryonic lethality in the case of homozygotes.

Therefore, we are proceeding to generate muscle creatine kinase (MCK)-Cre rats and myogenic factor 5(Myf5)-Cre rats for fukutin conditional knockout.

Soon we expect to have MCK-fukutin-cKO rats (dystrophic glycolysis model) and Myf5-fukutin-cKO rats.

If the transgenic rats mentioned above are produced, we will prepare to evaluate Fukutin's function and plan to report the results in a follow-up study in the CIMB journal.

We also corrected minor errors in the draft.

We request that you accept our paper for publication.

Round 3

Reviewer 2 Report

The authors should write a separate section about limitation of the study before the conclusion paragraph. Since the study objective is technical in nature and the transgenic rat is no more exist, the authors should modify the title and the objective of the manuscript. 

English language is fine.

Author Response

We are very grateful to reviewer 2 for his minor revision comments. We are very pleased to present a more complete paper thanks to Reviewer 2's careful review.

Reviewer 2

The authors should write a separate section about limitation of the study before the conclusion paragraph.

Response) Based on the reviewer's opinion, we wrote additional paragraphs at lines 234-238, the last part of the result, and lines 326-327, the last part of the discussion.

Since the study objective is technical in nature and the transgenic rat is no more exist, the authors should modify the title and the objective of the manuscript.

Since the purpose of our research is advancing transgenic technology and we continue to produce fukutin cKO rats, we have modified the title of the manuscript to "Optimizing embryo collection for application of CRISPR/Cas9 system and generation of fukutin knockout rat using this method".

Round 4

Reviewer 2 Report

The authors have addressed all my concerns in the revised manuscript. I support the publication of the manuscript.

Minor spelling mistakes are detected in the text.