In Silico Analysis of Pathogenic CRB1 Single Nucleotide Variants and Their Amenability to Base Editing as a Potential Lead for Therapeutic Intervention

Round 1

Reviewer 1 Report

Manuscript genes-1452758 “Analysis of pathogenic CRB1 single nucleotide variants and their amenability to base editing as a potential therapeutic intervention” by Bellingrath et al. The manuscript is the purely theoretical description of the potential of SNP correction using the Cas9 system. The authors made a substantial in silico work identifying the sites suitable for amendment in CRB1 variants. Manuscript well written and well presented. I would normally criticize the long introduction part, but it works quite well in the manuscript that also functions as a mini-review.

Comments: 

1. Please add your considerations on the challenges of delivery of the amendment constructs into the RPE cells, i.e., AAV proinflammatory effects in subretinal injections. Challenges when multiple corrections and thus constructs are required. Challenges associated with correction efficacy.  

2. As the authors encourage the scientific community to attempt actual SNP correction therapy with this manuscript, please present the sequence fragments of the amendable pathogenic SNP with the SNP site is marked as the supplementary table to simplify the development of the constructs by follow up in vitro and in vivo research. 

3. As the manuscript is purely theoretical sequence and SNP analysis authors encourage to soften the title "Analysis of pathogenic CRB1 single nucleotide variants and their amenability to base editing as a potential therapeutic intervention" to "In silico analysis of pathogenic CRB1 single nucleotide variants and their amenability to base editing as a potential lead for therapeutic intervention". Authors can use different wording but need to clearly indicate in silico and theoretical nature of the manuscript. 

Author Response

Response to Reviewer 1 Comments

 

Manuscript genes-1452758 “Analysis of pathogenic CRB1 single nucleotide variants and their amenability to base editing as a potential therapeutic intervention” by Bellingrath et al. The manuscript is the purely theoretical description of the potential of SNP correction using the Cas9 system. The authors made a substantial in silico work identifying the sites suitable for amendment in CRB1 variants. Manuscript well written and well presented. I would normally criticize the long introduction part, but it works quite well in the manuscript that also functions as a mini-review.

We greatly appreciate the time and effort the reviewer took to provide positive and helpful feedback on our article. We have used the reviewer’s comments to make adjustments and improvements to our submission. Please find a point-by-point response below.

Comments: 

Point 1: Please add your considerations on the challenges of delivery of the amendment constructs into the RPE cells, i.e., AAV proinflammatory effects in subretinal injections. Challenges when multiple corrections and thus constructs are required. Challenges associated with correction efficacy.

Response 1: The section 4.3 Further considerations in the article’s discussion has now been expanded to include comments on the translational challenges of a gene editing approach. As the reviewer suggest, topics such as the proinflammatory AAV effects, an all-in-one CRIPSR constructs and percentage correction efficiency for phenotype correction and are now reflected upon

 

 

Point 2: As the authors encourage the scientific community to attempt actual SNP correction therapy with this manuscript, please present the sequence fragments of the amendable pathogenic SNP with the SNP site is marked as the supplementary table to simplify the development of the constructs by follow up in vitro and in vivo research. 

 

Response 2: A supplementary table has now been added that lists all mutations amenable to base editing analysed in the paper. In addition to naming the base editor and PAM site for each mutation, the table contains additional information such as in which database the mutation can be found, the type of mutation, its clinical significance and the number of times listed in each publication. Instead of providing a sequence fragment, the exact location of each mutation is given by its cDNA position.

 

Point 3: As the manuscript is purely theoretical sequence and SNP analysis authors encourage to soften the title "Analysis of pathogenic CRB1 single nucleotide variants and their amenability to base editing as a potential therapeutic intervention" to "In silico analysis of pathogenic CRB1 single nucleotide variants and their amenability to base editing as a potential lead for therapeutic intervention". Authors can use different wording but need to clearly indicate in silico and theoretical nature of the manuscript. 

Response 3: The title has been changed according to the reviewer’s suggestion.

 

Author Response File: Author Response.docx

Reviewer 2 Report

In this paper the authors review over a thousand of CRB1 mutations and analyzed them for their amenability to DNA base editors and available PAM sites from a selection of Cas proteins. Through the evaluation of ClinVar and LOVD databases, authors listed all reported SNVs in CRB1. Each SNV is then characterized according to reported classification (pathogenic to benign) in databases, exon location, editability, and mutational consequence. Furthermore, an evaluation of amenability of pathogenic SNVs to base editing has been conducted. Authors selected appropriate base editor and PAM sites in order to provide an indication of the feasibility of gene editing for CRB1-associated retinal degeneration.

The argument is interesting, the large review and characterization of SNVs in CRB1 can be a solid base to develop research studies on base editing. In my opinion, it is a very good paper, well written and well represented. Just a suggestion that, in my opinion, can improve the paper and support the characterization of SNVs. Authors say that “Since the gold standard ACMG guidelines for variant classification were published in 2015, a review of mutations assessed before that time point may greatly improve the dataset quality and unify the classification criteria applied for mutation within and between datasets.” In the paper, conflicting pathogenicity SNVs are further stratified in pathogenic or benign according to the most frequent report. However, an evaluation according to ACMG guidelines could help in re-classification of variants that can not be stratified because reports are very conflicting (benign VS pathogenic). I know that some ACMG criteria requires clinical or familial data (unavailable in ClinVar and LOVD dataset), but I think that authors should consider performing a re-evaluation of very conflicting variants according to ACMG guidelines.

 

Author Response

Response to Reviewer 2 Comments

 

In this paper the authors review over a thousand of CRB1 mutations and analyzed them for their amenability to DNA base editors and available PAM sites from a selection of Cas proteins. Through the evaluation of ClinVar and LOVD databases, authors listed all reported SNVs in CRB1. Each SNV is then characterized according to reported classification (pathogenic to benign) in databases, exon location, editability, and mutational consequence. Furthermore, an evaluation of amenability of pathogenic SNVs to base editing has been conducted. Authors selected appropriate base editor and PAM sites in order to provide an indication of the feasibility of gene editing for CRB1-associated retinal degeneration. The argument is interesting, the large review and characterization of SNVs in CRB1 can be a solid base to develop research studies on base editing. In my opinion, it is a very good paper, well written and well represented.

We greatly appreciate the time and effort the reviewer took to provide positive and helpful feedback on our article. We have considered the reviewers excellent point and have amended our article. Please find a detailed response below.

Comments: 

Point 1: Just a suggestion that, in my opinion, can improve the paper and support the characterization of SNVs. Authors say that “Since the gold standard ACMG guidelines for variant classification were published in 2015, a review of mutations assessed before that time point may greatly improve the dataset quality and unify the classification criteria applied for mutation within and between datasets.” In the paper, conflicting pathogenicity SNVs are further stratified in pathogenic or benign according to the most frequent report. However, an evaluation according to ACMG guidelines could help in re-classification of variants that can not be stratified because reports are very conflicting (benign VS pathogenic). I know that some ACMG criteria requires clinical or familial data (unavailable in ClinVar and LOVD dataset), but I think that authors should consider performing a re-evaluation of very conflicting variants according to ACMG guidelines.

Response 1: The reviewer makes an excellent point about the challenge of dealing with conflicting mutations in the analysis dataset. The reviewer also provides a very relevant point on re-classifying the conflicting mutations assessed before 2015 according to the ACMG guidelines. The classification and inclusion of conflicting mutations into our dataset is certainly a challenge we grappled with extensively in preparation for writing this manuscript. While we did consider re-classifying the conflicting mutations, we ultimately decided against this, since we felt an expert consortium of geneticists was needed to draw such a conclusion. We were also aware of the potential consequence of incorrectly labelling a mutation as pathogenic. In an effort to present the data in the most transparent way possible, we included the conflicting variants that had been labelled pathogenic/likely pathogenic at least once, all while retaining the label of “conflicting pathogenic”.

To address the reviewer’s well-made point of including information on the date of the last mutation evaluation, we have provided a supplementary table that lists all the editable mutations analysed in this dataset. In addition to information like mutation location, mutation consequence and clinical significance, we have provided a date when each mutation was last reviewed, provided this was information given in the datasets. With exception of 3 conflicting mutations, all conflicting mutations included were evaluated after 2015. We very much agree with the reviewer that the evaluation of conflicting mutations by an expert consortium is needed.

 

Author Response File: Author Response.docx

Reviewer 3 Report

The article by Bellingrath et al. have reviewed all the single nucleotide variants reported for CRB1 gene from two different databases- LOVD and ClinVar. They have further investigated the availability of these SNVs for different DNA base editors and also looked for potential PAM sites in those regions. 

While it would have been great to see some actual targeting of some of the SNVs reported in this article using CRISPR-Cas, nevertheless this article provides a great starting point for anyone who wants to pursue gene editing for CRB1 mutations in future.

Author Response

Response to Reviewer 3 Comments

 

The article by Bellingrath et al. have reviewed all the single nucleotide variants reported for CRB1 gene from two different databases- LOVD and ClinVar. They have further investigated the availability of these SNVs for different DNA base editors and also looked for potential PAM sites in those regions. 

While it would have been great to see some actual targeting of some of the SNVs reported in this article using CRISPR-Cas, nevertheless this article provides a great starting point for anyone who wants to pursue gene editing for CRB1 mutations in future.

We would like to thank the reviewer for their time and effort in evaluating our article. We greatly appreciate the positive and encouraging feedback on our article.

 

Author Response File: Author Response.docx