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The authors have addressed all comments. I support the publication of the manuscript.
[# PeerJ Staff Note - this decision was reviewed and approved by Keith Crandall, a PeerJ Section Editor covering this Section #]
The author should take into account all comments and recommendations of the reviewers to improve this work.
[# PeerJ Staff Note: This submission is a _Stage 1 Registered Report_. This is a new initiative at PeerJ and the review criteria are slightly different from a regular research article. #]
• The introduction and background nicely presented and relevant literature cited. The sentence describing the study objective is too long. Plz simplify it and if possible divide it into two sentences. (line # 91-93)
• The authors have listed various freely available softwares for in silico analysis for detection of mutations in the existing CRISPR-Cas-based diagnostic assays. However, considering the constant addition of SARS-CoV-2 sequences from around the globe, I highly recommend the authors to make a web-based tool. The user interface of this web tool incorporates all the free softwares enlisted/required in the assessment of mutations. This will allow clinicians and researchers to quickly assess the presence or absence of mutations in target regions of any future diagnostic assay.
• Alternatively, to automate the pipeline for basic scientists and clinical researchers the authors can make their methods pipeline in the form of a script to be run using command line (cmd for windows/Terminal for mac).
• The analysis part seems quite laborious and require manual inspection of thousands of sequences.
o The sequence alignment can be easily replaced by ‘R’-based script which performs the sequence alignment (‘msa’ package). The script can also include code for inspection of sequences and determine the variability. Furthermore, PNNS can also be incorporated in the script. For more information please read the following links.
https://www.bioconductor.org/packages/release/bioc/vignettes/DECIPHER/inst/doc/ArtOfAlignmentInR.pdf
https://bioconductor.org/packages/release/bioc/vignettes/msa/inst/doc/msa.pdf
https://cran.r-project.org/web/packages/ggmsa/vignettes/ggmsa.html
Not applicable at this time.
In my professional opinion the authors should automate their methods either by making a web-based portal or making an 'R'-based script. However, if they feel that at this time they do not have the expertise or time to make an automated solution, the current methodology is totally acceptable for the purpose of publication.
This manuscript proposes a bioinformatic pipeline for analyzing the specificity of CRISPR gRNAs used in CRISPR-based SARS-CoV-2 diagnostics. However, no actual sequence analysis is performed and no results are obtained and no original data presented. This manuscript would fall under the category of "Hypothesis Papers".
Not applicable - the manuscript does not present original results.
Not applicable - the manuscript does not present original results.
Some suggestions:
1. This manuscript proposes to assess the on-target specificity of CRISPR gRNAs used for SARS-CoV-2 diagnostics. This bioinformatic analysis could be improved by incorporating a counter selection against sgRNAs to exclude those that cross-react with other viral, human or bacterial genes.
2. Line 80-83: The author states, “One of the focus of CRISPR-diagnostics remained to improve assay specificity as tolerance of mismatches by Cas9 can result in a false-positive diagnosis. This risk has been minimized with the newer Cas proteins, Cas12 and Cas13, that have a lower tolerance for mismatches compared to Cas9 especially in the “seed” region.” .
This is not accurate — Cas9 is not used in CRISPR diagnostics because, unlike Cas12 and Cas13, Cas9 does not exhibit non-specific trans-activity upon target sequence binding. It is the trans-activity of Cas12 and Cas13 that enable these proteins to trigger a detectable signal upon target sequence binding/identification.
3. Line 58: the author gets the nomenclature between Cas12a and Cas13a incorrect. Cas12a = Cpf1, Cas13a = C2c2.
4. Line 15: what is a stage 1 protocol?
no comment
no comment
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The author is going to use the protocol he used already for qPCR test systems. He made corrections accordingly to suit the new test target and offered the new design to incorporate a broader virus sequence set. Overall, the intro is sound. There was one technical typo: in line 58, Cas12a, and Cas13a former names are mixed up. Author might want to add the reference to another Cas13a-based assay CARMEN by Ackerman et al., Nature, 2020 (nature.com/articles/s41586-020-2279-8).
Although comparing the protospacer to the alignment is more straightforward than comparing three-component qPCR oligo sets, several concerns should be addressed:
1 PAM may have degenerate bases by its nature;
2 PAM length may vary from one-two bases to six-seven for different Cas proteins and mutant derivates thereof (https://www.addgene.org/guides/crispr/#rna-targeting);
3 PAM orientation is relevant to protospacer;
4 Seed regions, as pointed by author, differ for Cas proteins, so the influence of mismatch position and mismatch count varies;
5 Different Cas proteins tolerate mismatches differently;
6 SNP frequency should be assessed correctly after batch alignments concatenation.
As it is only planned to study around a dozen of protospacers, all these questions can be easily addressed.
“More than one mismatch” threshold offered by Bioinformatics Institute for CoVsurver author is planning to use in this study might be to broad. If 60000 isolates would be used, two sequences with the same mismatch will represent only 0.003% of the set. For CoVsurver, authors use a threshold of more than one functional mutation, i.e., leading to an amino acid substitution. Variation type does not play a role for Cas-based test purposes. This strict threshold may lead to wrong conclusions.
From the Cas-based SARS-CoV-2 detection assays designer standpoint, it should be mentioned, that they had small maneuver space, as they should find protospacer common to all SARS-CoV-2 strains, yet unique to SARS-CoV-2 coronavirus, and Cas-specific PAM should be adjacent to the target region. Compared to a qPCR test, for Cas-based tests, specificity depends only on one oligo strain of approximately 20 bases, but on the other hand, it makes Cas-based tests less prone to mismatches.
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