Transcriptomic Investigation in CRISPR/Cas9-Mediated GRIK1-, GRIK2-, and GRIK4-Gene-Knockout Human Neuroblastoma Cells

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This is a well written manuscript that addresses whether glutamate ionotropic kainate receptors encoded by the GRIK gene family are implicated in the pathophysiology of schizophrenia.  The manuscript is timely and adds to our understanding of how kainate receptors could play a role in the development of schizophrenia.  The CRISPR/Cas9 genome editing system was used to create isogenic candidate receptor gene deficiencies in SH-SY5Y cell lines. 

General concerns:   

1. The authors sometimes overstate the implications of their findings.  For example, in the last sentence of paragraph 3.2, it states “these findings suggest that kainate receptor-regulated synaptic membrane genes are implicated in synaptic dysfunction in the pathophysiology of schizophrenia”.  While it is possible that kainate receptor-regulated synaptic membrane genes serve a role in the synaptic dysfunction of the pathophysiology of schizophrenia, the present findings are not definitive.  It would be best to change the statement to say that they “could possibly be implicated”. 

2. Similarly, the authors date in the last sentence section 3.3, that the findings suggest that kainate glutamate receptors regulate actin-based cytoskeleton, which is essential for maintaining in diuretic spine morphology intensity in the pathophysiology of schizophrenia.”  The findings in cell culture do suggest that kainate glutamate receptors play an important role in actin-based cytoskeleton production, but that does not mean that they have a regulatory role. 

 

Specific concerns:

1. On page 9, in the last sentence, it should state "we suggest" instead of " we suggested".

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript is very interesting from both fundamental and practical points of view. The authors used the CRISPR/Cas9 system to create knockouts on the GRIK1, GRIK2 and GRIK4 genes encoding kainate receptor subunits in the SH-SY5Y line and proposed a mechanism linking mutations in these genes to the development of schizophrenia. However, some points require further clarification.

 

It would be good to make line numbering in the template for the journal, that way it's easier to indicate comments.

 Please make the title of the paper more informative. In this paper, you are not investigating a single gene knockout line (as can be understood from the title), but several lines with knockout of three kainate receptor genes, and you are focusing on studies of those differentially expressed genes that are associated with the pathogenesis of schizophrenia.

 Please include in the keyword list schizophrenia and the names of kainate receptor genes.

 You indicated that the rare mutations you found in humans are GRIK1p.Phe24fs, GRIK1p.Thr882fs, GRIK2p.Arg300Ter, and GRIK4p.Gln342Ter. At the same time, you used the lines GRIK1p.L25Pfs*?/p.L25Pfs*?, GRIK2p.L301Ffs*?/p.L301Ffs and GRIK4 p.H343Afs*?/p.H343Afs*? Please explain why you chose to mutate neighboring amino acids rather than reproduce exactly the previously discovered mutations?

 Figure1. In the legend to Figure 1, replace "two edited cells" or "one edited cells" with "two edited cell lines" or "one edited cell lines" respectively.

 The SH-SY5Y cell line is derived from a cancer and it would be more convincing to show the validity of the cell models you obtained by enrichment analysis for the disease-associated genes among DEGs based on the DisGeNET database. For example, as done in Figure 5A in the article https://www.mdpi.com/1422-0067/24/21/15746.

 Figure 2. It would be more representative to add hierarchical clustered heatmaps (e.g., as in Figure 1 in the article https://www.frontiersin.org/articles/10.3389/fcell.2020.00310/full) to visualize the differences and similarities in groups of differentially expressed genes between the mutated lines and the original line.

 Please enlarge the font of the labels in Figure 2. In the current form they are unreadable.

You have indicated the number of DEGs and the pathways where statistically significant changes in GO terms were observed in the mutants compared to WT cells. However, the direction and extent of these changes are not indicated. Please provide a more detailed and interpretable functional analysis of the transcriptome and analyze the GO and KEGG enrichment of DEGs. This would provide a better understanding of exactly how and to what extent pathway activity is altered in different mutants and allow more accurate conclusions to be drawn. As an example, see Figure 2 in the article https://www.mdpi.com/2073-4425/11/2/187

 At the beginning of section 2.4, please add a brief explanation of why the structure of the cytoskeleton was investigated.

 According to the qRT-PCR data shown in Figure 3, GRIA4 knockout causes an increase in the expression of most genes, including its own.

 What possible explanation can the authors offer for this effect? Are there any transcription factors among the differentially expressed genes, that could explain the observed changes?

 Based on the available and additional results, please draw a simple diagram of possible biochemical, signaling, or other pathways that could link mutations in the GRIK1, GRIK2, and GRIK4 genes to abnormalities in membrane-bound gene expression and/or cytoskeleton associated with schizophrenia? This will make it much easier for readers to understand the conclusions of this paper.

 Section 4.2. Please indicate which statistical test was used and what correction for multiple comparisons was used.

 Please correct the conclusions according to the new data obtained with additional analyses of the transcriptome data.

 Supplementary Data.  You stated that "Considering the off-target effects of the CRISPR/Cas9, we confirmed with Sanger sequencing and revealed null off-target results in each edited cell line". If you have any Sanger sequencing results left over, could you add them in the Supplementary files?

Please add lists of differentially expressed genes (between mutants and wild type) with degree of change and p-value in the Supplementary Data.

Comments on the Quality of English Language

English is good.

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have greatly improved the manuscript by responding to all comments. I have no further significant comments and suggest that the manuscript be accepted for publication in the journal.