SciScore for 10.1101/2021.06.06.446826: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Table 2: Resources

Results from OddPub: Thank you for sharing your data.

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: We did not find any issues relating to colormaps.

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

Results from scite Reference Check: We found no unreliable references.

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

new tag?:

• extract reusable functions to reduce duplication / allow elegant patterns elsewhere

new tag?:

• understand why it is correct

SciScore for 10.1101/2021.06.04.447160: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Table 2: Resources

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

Results from JetFighter: We did not find any issues relating to colormaps.

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

Results from scite Reference Check: We found no unreliable references.

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

SciScore for 10.1101/2021.06.02.21258073: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Table 2: Resources

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: We did not find any issues relating to colormaps.

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a protocol registration statement.

Results from scite Reference Check: We found no unreliable references.

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

Same feedback as the Atelier. In addition,

• The job title and "self-employed" sound inconsistent. Consider a more powerful title. I'd get rid of the self-employed tag. It's got a bit of a bad reputation on LI
• The company description needs to add some points of differentiation. Currently it doesn't impress.

SciScore for 10.1101/2021.06.02.446468: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Table 2: Resources

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: We did not find any issues relating to colormaps.

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

Results from scite Reference Check: We found no unreliable references.

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

SciScore for 10.1101/2021.05.26.21256092: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Table 2: Resources

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: We did not find any issues relating to colormaps.

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

Results from scite Reference Check: We found no unreliable references.

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

Should this be in an annotations property of the Canvas, Manifest, or AnnotationPage?

Reviewer #1 (Public Review): 

The paper by Sim et al describes phospho-proteomic analysis of ATR kinase-dependent pathway in mouse spermatocytes. By administrating an ATR inhibitor, AZ20, to mice and using Rad1 (a component of 911 DNA damage clamp) conditional knockout mouse (cKO), the authors isolated testis from these mice, isolated phospho-peptides and analyzed with Tandem Mass Tag (TMT). The analyses identified 37,180 phosphorylation sites and created the data base for them. Importantly, in-depth analysis of the phosphorylation sites revealed an unique consensus site of the ATR-dependent phosphorylation; S/TPXK, whose kinase has not been identified yet. In addition, the authors showed new ATR-dependent phosphorylation sites in proteins in RNA metabolisms including piRNA biogenesis for transposon silencing and showed ATR-dependent localization of two RNA processing enzymes, SETX helicase, and RANBP3 in meiosis sex chromosome inactivation (MSCI). This is an important body of work as a good resource for phosphorylation in mouse germ cells. The study had been done in a great care with proper controls. The data set in the paper is very much useful and of great interest to researchers in meiosis and DNA damage response (DDR) field.

SciScore for 10.1101/2021.05.26.21257441: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Table 2: Resources

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

While our study aims to provide an overview of many different aspects of pre-existing immunity against VOC, there are some limitations. In this study, we present a vaccinee population that consists of primary health care workers that are generally of middle age, with only four (8%) participants being older than 60 (Table 1). However, we found no correlation between age and titers in convalescent patients against any of the VOC tested. Moreover, we have focused on the neutralization potential of immune sera and did not examine antibody effector functions which have been implicated previously in the control of SARS-CoV-2 infection (41). Similar to other studies, our convalescent and vaccinee sera samples were collected four to six weeks after infection and four weeks after vaccination (i.e. at the peak of immunity), respectively. While we present findings that may have implications for additional booster vaccines and the monitoring of VOC, we did not examine the aspect of waning antibody titers and how those might influence VOC binding and neutralization. Finally, we have merely examined an early line of defense against infection with SARS-CoV-2 (i.e. serum antibody levels), while we expect memory B cells to play an important part in re-infections with SARS-CoV-2 VOC. Indeed, swift re-activation of memory compartments may lead to reduced transmissibility, a milder course of disease and a more potent immune response. In conclusion, we observed a substantial reduction in binding ...

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: We did not find any issues relating to colormaps.

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a protocol registration statement.

Results from scite Reference Check: We found no unreliable references.

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

SciScore for 10.1101/2021.06.01.446555: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Table 2: Resources

Results from OddPub: Thank you for sharing your data.

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: We did not find any issues relating to colormaps.

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

Results from scite Reference Check: We found no unreliable references.

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

• not worth saying
• not necessary to say/write
• useless information

Suppress-Script

unstated-but-realized

Why was it not free???

select ... over

database: query builder

like ActiveRecord for node

SQL: experimenting with table-free data

SciScore for 10.1101/2021.05.29.446300: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Table 2: Resources

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

At present, the limitations for selecting POI for Antibody Display such as protein shape and size remain unclear; further studies will be required to address these questions. Comparing with conventional Fc fusion proteins in which the POI is fused to the N terminus of Fc usually including the flexible hinge region, we reasoned that the Antibody Display, which grafts the POI onto the β-strands G and F of the heavy chain, provides several potential advantages: (1) design and production of a more conformationally constrained chimeric protein, (2) reduction of proteolysis with the POI inserted into a stable antibody Ig fold, and (3) extension of the current antibody engineering toolkit for generating bispecific and chimeric antibodies. Further studies will be needed to explore these potential advantages. An intriguing feature of using the Antibody Display for Tspan12EC2 is with a better yield of protein production than Tspan12EC2. Our functional assays demonstrated that Tspan12EC2 bound to Norrin directly, in agreement with previous genetic studies (Lai et al., 2017). As Tspan12 playing critical roles in CNS vascular development (Junge et al., 2009; Zhang et al., 2018) and tumorigenesis (Knoblich et al., 2014; Otomo et al., 2014), Tspan12EC2-AD may be a useful reagent for these studies. Moreover, Tspan proteins are well known for their important roles in regulating tumor migration, invasion and metastasis (Charrin et al., 2014; Hemler, 2014; Vences-Catalan and Levy, 2018). Antibo...

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: We did not find any issues relating to colormaps.

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

Results from scite Reference Check: We found no unreliable references.

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

SciScore for 10.1101/2021.05.31.445667: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Table 2: Resources

Results from OddPub: Thank you for sharing your data.

Results from TrialIdentifier: We found the following clinical trial numbers in your paper:<br><table><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Identifier</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Status</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Title</td></tr><tr style="background-color:#FF0000"><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NCT811</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Trial number did not resolve on clinicaltrials.gov. Is the number correct?</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NA</td></tr></table>

Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

Results from JetFighter: We did not find any issues relating to colormaps.

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

Results from scite Reference Check: We found no unreliable references.

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

SciScore for 10.1101/2021.05.31.446386: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Table 2: Resources

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on page 28. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

Results from scite Reference Check: We found no unreliable references.

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

SciScore for 10.1101/2021.05.28.446179: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Table 2: Resources

Results from OddPub: Thank you for sharing your data.

Although several articles and preprints in the past year74,79–84 have reported simulations of this structure, a review of their results shows similar limitations, with abbreviated timescales,80–82 reliance on secondary-structure restraints,74 elevated RMSDs,74,80 and/or dewetting in the absence of electric fields.74 Instability of the open structure may reflect underdetermination of the starting protein or membrane models, unresolved interactions with the C-terminal or other domains, or other factors yet to be identified. Taken together, we have shown that recombinantly expressed full-length E protein from SARS-CoV-2 is capable of independent multimerization, possibly as a tetrameric or smaller species. We also confirmed that the protein localizes intracellularly, similar to its predecessor from SARS-CoV, with no evidence of ion channel properties at the cell surface. Our coarse-grained simulations further support a role for the E protein in viral budding, as the presence of the protein bends the surrounding membrane. Reduction of intracellular Ca2+ in E-protein-transfected cells may further promote viral replication. However, our atomistic simulations and permeation calculations based on previously reported NMR structures resulted in unstable proteins incapable of Ca2+ conduction. We emphasize the importance of using high-resolution structural data obtained from a full-length protein to gain detailed molecular insight of the E protein, and enable future drug-screening effort...

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: We did not find any issues relating to colormaps.

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

Results from scite Reference Check: We found no unreliable references.

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

• supporting those you like by sending business to them
• less formal and more approachable

.

SciScore for 10.1101/2021.05.26.445185: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Table 2: Resources

Fitting was carried out in MATLAB by non-linear least-squares regression using the Trust-Region algorithm.</td><td style="min-width:100px;border-bottom:1px solid lightgray"><div style="margin-bottom:8px"><div>MATLAB</div><div>suggested: (MATLAB, RRID:SCR_001622)</div></div></td></tr></table>

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

Results from JetFighter: We did not find any issues relating to colormaps.

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

Results from scite Reference Check: We found no unreliable references.

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

art factores

definicion referencia oportuna

I've noticed that Dan Mackinlay has some public notebooks with an interesting system for indicating knowledge process too.

His Twitter tag line is: "The Spectator's Wiki Man."

There are a number of bloggers who have to some extent, specifically used their blogs for this purpose though. I've documented several at https://boffosocko.com/tag/thought-spaces/

cherry-picking only some commits from a PR

where did it come from? don't remember

after a while, something that came from another starts to feel like your own

you make it your own

happy middle/medium?

I wonder if this insulation will be applicable to other gram negatives? (or other gram positives too?)

SciScore for 10.1101/2021.05.21.445201: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Table 2: Resources

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

Results from JetFighter: We did not find any issues relating to colormaps.

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

Results from scite Reference Check: We found no unreliable references.

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

SciScore for 10.1101/2021.05.23.445348: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Table 2: Resources

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: We did not find any issues relating to colormaps.

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• No funding statement was detected.
• No protocol registration statement was detected.

Results from scite Reference Check: We found no unreliable references.

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

This as since been renamed to updateless decision theory%20is,which%20it%20makes%20its%20decisions.&text=It%20acts%20as%20though%20its,some%20dualist%20free%2Dwill%20theories.)

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

Referee #1

Evidence, reproducibility and clarity

Summary:

In the manuscript „Intra-helical salt bridge contribution to membrane protein insertion" the authors investigate the effect of salt bridge formation between positively and negatively charged amino acids on the insertion behavior of α-helical protein segments into the membrane. Generally it is believed that polar or even charged residues prevent stable membrane insertion of α-helical protein segments, but some of these authors had already shown in a previous paper that such residues are more frequent than expected in transmembrane helices. In the current study, the authors investigate in detail the role of intra-helical salt bridge formation on stable membrane insertion. Using an in vitro membrane insertion assay based on the E. coli leader peptidase (Lep) protein, they found better membrane insertion for helical segments with opposite charge pairs placed at positions compatible with intra-helical salt bridge formation (positions i→i+1; i→i+3 and i→i+4). Furthermore, the authors performed a database screen which revealed that oppositely charged residues are overrepresented at these positions. Finally they picked two candidate membrane proteins from the database (Halorhodopsin and calcium ATPase 1) and proved the presence of an intra-helical salt bridge and determined the contribution of the salt bridge to the apparent free energy of membrane insertion (ΔGapp), which was in the range of 0,5-0,7 kcal/mol.

Major comments

1. It seems that the data in Fig. 3b has been mixed up, making it difficult to judge the conclusions. The bars with forward slash seem to represent the "same charge" data and the bars with backward slash seem to represent the "opposite charge" data (exactly contrary to the figure legend). In general the forward and backward slash representation is not easily distinguishable, and for the position i+4 both bars contain a forward slash (making it impossible to discriminate same and opposite charge). Please use filled and unfilled bars instead. Furthermore the bar diagram in Fig. 3a is stacked for opposite and same charge, whereas in Fig. 3b the respective bars are placed next to each other. Additionally the label of the y-axis in Fig. 3c is misleading, as it is not the "Frac. of opp. charged pairs" but the fraction of oppositely charged pairs that form salt bridges.
2. The authors don´t give details no how the log odds ratios and the respective p-values have been determined. Please include this in the Materials and Methods section. What does a p-value of 0.00e+00 mean (see Table 2, Spacing: +3, "All Log odds")?
3. What is the proof that for the isolated helix A from the calcium ATPase 1 the membrane embedded part is identical to the full-length protein? The authors investigated two different helix A peptides, the full-length helix ranging from L49-F78, and one short fragment ranging from L49-A69 containing the more hydrophilic N-terminal region, which is the membrane-embedded region in the full-length protein. The authors state: "In contrast, when only the membrane-embedded sequence was included, the Lep chimera was mainly doubly-glycosylated (Fig. 5c, lane 3), suggesting that helix A is properly inserted when the full helical sequence is present." In my opinion this conclusion cannot be drawn from the data presented. The authors used an isolated helical segment, so in my opinion it is much more likely that the isolated full-length helix inserted via its hydrophobic C-terminal part (L60-F78) into the membrane. The authors themselves state in their manuscript: "It has been previously shown that the position in the membrane of TM helices in protein folded structures does not always correspond to the thermodynamically favored positions in the membrane of the isolated helices." Also the i→i+5 mutant points into that direction, because the effect of disturbing the intra-helical salt bridge for the helix A is much less pronounced compared to the similar data in Fig. 4f for the Halorhodopsin protein. In my opinion this shows that most probably only one charged residue (R63) is embedded inside the membrane (with a membrane embedded part of L60-F78).

Minor comments:

1. line 151: ",see Figure 2)" Typo: Bracket missing.
2. line 172: "Other known structural features can also be hinted at, including aromatic ring stacking by His-Trp pair [20] at i→i+6." Please give some more examples of important structural features of membrane proteins, which can be seen in your analysis (e.g. I think that also the glycine zipper can be seen in the i→i+4 data set).
3. line 255: "The salt bridge contributes approximately ~0,5 kcal/mol to the apparent experimental free energy of membrane insertion." Please explain that this value was derived from the comparison of the ΔGexp between the wt and the i→i+5 mutant. Please comment also on the large difference between the predicted (ΔGpred) and the experimental values (ΔGexp), even if no salt-bridge is involved (e.g. for the DD mutant).
4. line 348: "Asp-Lys pairs at position i, i+4 and Glu-Lys pairs at position i→ i+3 are the most prevalent as seen previously in Figure 2. They are both among the most prevalent oppositely charged pairs and the charged pairs that form the highest number of salt bridges in membrane protein structures. This is in stark contrast to Glu-Arg pair at position i→ i+1 that although as frequent in pairs as Asp-Lys and Glu-Lys at positions i→i+4 and i→i+3 respectively, only form salt bridges in one-fourth of the cases." Fig. 2 shows that each charge pair has a different prevalence depending on the order (e.g. Asp-Lys and Lys-Asp pairs). I think for this statement the sum of both prevalences should be taken into account, and as the sum is not easy discernible from Fig. 2, it would help to include a table containing the sums. Furthermore, it would be good to refer also to Fig. 3, which also contains a part of the discussed data.
5. line 402: "c-myc tag (Glu-Gln-Lys-Leu-Ile-Ser-Glu-Glu-Asp-Leu, EQKLISEEDL) was added in Ct in hanging with de PCR primer before cloning." Please revise the sentence and I think the one letter code for the c-myc tag is sufficient (please correct this also in line 428).
6. line 420: "A region's total ΔG is the sum of these individual scores weighted on where in the region the residue, a residue in the middle of the helix has a higher weight than residues at the ends." Please revise the sentence, the meaning is unclear.
7. line 436: "Total protein was quantified and equal amounts of protein submitted to Endo H treatment or mock-treated, followed by SDS-PAGE analysis and transferred into a PVDF transfer membrane (ThermoFisher Scientific)." Please revise the sentence.
8. line 498: "Topological files with sequence and membrane topology are created with the help of the RCSB secondary structure file and only membranes annotated as pure α-helices were retained." I assume that the description contains a typo (membranes annotated as pure α-helices?)
9. line 507: typo "..., but we did not clustered the proteins" 14: line 560: "The individual value of each experiment in represented by a solid dot being represented as a green square the experimental ΔG value for the L4/A15 sequence from [2]." Please revise the sentence. 15: line 562: "The wt and simple mutants are shown in white bars." Typo: single mutants 16: line 563: "Charges at compatible distances with salt bridge formation (i→i+1; i→i+3; and i→i+4) are shown in yellow. Not compatible distances with salt bridge formation (i→i+2; and i→i+5) are shown in dark gray. Compatible distances but not compatible amino acid pair (i, i+4 DD pair) is shown in clear gray." The given colors don´t match with the figure (i→i+1 = brown; i→i+3 = orange; i→i+4 = yellow and i→i+4 DD pair = white) 17: line 597: "The different monomers are shown in transparent blue, purple and indigo." The different colors are hardly distinguishable in the figure. 18: Figure 4a: The table could be simplified. I think the column "charges" can be removed, as it contains not really charges and the names of the peptides already contain the same information. The column "Å" contains only a value for the wt (and not for the DK i,i+5 mutant) and as the distance for the wt is also given in Fig. 4g, this column can be also removed.
10. Fig. 4f: The marker lane is hardly visible (completely dark lane)
11. Fig. 5b: The column "Å" contains only values for the wt sequences (long and short). See also comment 18.
12. Fig 5d: Why is in the SDS gel a mass shift between the wt and the i→i+5 mutant visible, even though the peptide mass is equal.
13. There are several changes of font type or format changes (e.g. line 210-214). Please correct this.

Significance

As a structural biologist with a focus on membrane-proteins, I understand that the study is concerned on intra-helical salt bridges, but the implications of inter-helical salt bridges should also be discussed, at least in the introduction or outlook. The authors propose that their results are important for the improvement of membrane protein topology prediction methods, so for this aim it is also necessary to take any potential inter-helical salt bridges into account. In this context, it would be relevant to point point out that there even exist extended rows of salt bridges between transmembrane segments (charge-zippers), which serve an important structural element in several membrane proteins.

The article is well written and most of the conclusions drawn from the experimental results are convincing. I agree with the authors that their results are relevant for future improvement of membrane protein topology prediction software, which so far does not take the possibility of salt bridge formation into account. Therefore, I recommend publication after clarification/revision of the abovementioned points.

SciScore for 10.1101/2021.05.19.444889: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Table 2: Resources

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on pages 10 and 21. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

Results from scite Reference Check: We found no unreliable references.

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

SciScore for 10.1101/2021.05.16.444369: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Table 2: Resources

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: We did not find any issues relating to colormaps.

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

Results from scite Reference Check: We found no unreliable references.

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

SciScore for 10.1101/2021.05.15.444222: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Table 2: Resources

Results from OddPub: Thank you for sharing your code and data.

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: We did not find any issues relating to colormaps.

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

Results from scite Reference Check: We found no unreliable references.

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

SciScore for 10.1101/2021.05.13.444010: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Table 2: Resources

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: We did not find any issues relating to colormaps.

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• No funding statement was detected.
• No protocol registration statement was detected.

Results from scite Reference Check: We found no unreliable references.

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

IMHO #go == [[go]]. In that sense we should probably support all conventions that clearly demonstrate an [[intent]] to trigger an action, I'm fine adding support for these.

DOI: 10.1016/j.celrep.2021.109151

Resource: (Thermo Fisher Scientific Cat# 26183, RRID:AB_10978021)

Curator: @Naa003

SciCrunch record: RRID:AB_10978021

Curator comments: HA Tag Monoclonal Antibody (2-2.2.14) Thermo Fisher Scientific Cat# 26183

What is this?

This is an example of spaced repetition being done as retrospective and hiding some of the value of making the important things stand out and reviewing them for better long term retention.

Tag:RSS,ColorAposematico

https://pubmed.ncbi.nlm.nih.gov/rss/search/1-7FJ7kkHKrEcV2x8GNHBU9h3RdKSg9-Zlru1eJQISJRXlEg5e/?limit=100&utm_campaign=pubmed-2&fc=20210518104625

https://twitter.com/bugsnstuffcom/status/1301445545299505153

Tag:ColorAposematico

https://www.wikidata.org/wiki/Q619751

"Tag:"ColorAposetamitco

TAG:CicloVida

Tag:Colage

**Tag:"Aposematismo,ColorAposematico,Query

Tag:Aposematismo,ColorAposematico,Query

Tag:Aposematismo,ColorAposematico,Query

https://doi.org/10.1111/jeb.12676 TagColorAposematico

https://doi.org/10.1093/beheco/arl046

Tag:ColorAposematico

https://doi.org/10.1007/BF00384948

Tag:ColorAposematico

https://doi.org/10.1016/j.tree.2005.07.011

Tag:ColorAposematico

https://doi.org/10.2307/3544135

Tag:ColorAposematico

SciScore for 10.1101/2021.05.06.21256766: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Table 2: Resources

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Regarding the potential limitations, we did not collect skin swabs to control for the potential presence of SARS-CoV-2 in the skin [7,33]. However, all milk samples as well as the infants were negative for SARS-CoV-2 presence. Thus, although there are still limited data, it is accepted that breastfeeding does not represent a vehicle for vertical transmission of SARS-CoV-2 [9]. There are still many open questions: when are SARS-CoV-2 antibodies produced after maternal infection, when can they be detected in breast milk, and how long do they persist. To cover some of these questions, we aimed to determine the presence of antibodies in breast milk samples from COVID-19 women and to compare these with milk samples collected prior to the pandemic as reference controls. While different studies have reported presence of specific IgA antibodies against SARS-CoV-2 [7,12,13,36], limited information is available on IgG and IgM. Our results showed presence of anti-SARS-CoV-2 antibodies in milk, primarily IgA but also IgG and IgM targeting RBD. Furthermore, IgA- and IgG against non-structural MPro were analyzed for the first time in human milk samples. High intra- and inter-individual variability was observed in antibody presence and significant differences were observed for all three antibody classes when compared to prepandemic samples in agreement with previous data [26]. In our dataset we found that 82·9 % of the milk samples tested positive for the RBD antigen at least in one of the ...

Results from TrialIdentifier: We found the following clinical trial numbers in your paper:<br><table><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Identifier</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Status</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Title</td></tr><tr><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NCT04768244</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Recruiting</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Impact of Maternal COVID-19 Disease on Breast Milk and Infan…</td></tr><tr><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NCT03552939</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Completed</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">The Impact of Maternal Microbes on Infant Health Programming</td></tr></table>

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: We did not find any issues relating to colormaps.

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

Results from scite Reference Check: We found no unreliable references.

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

这段主要解释嵌套匿名结构在 marshal or unmarshal 时，field key 的冲突情况。我把 untagged 域 json 化的 key 称为 field key，tagged 域 json 化的 key 称为 tag key。

注意：这里讲的 tagged 域是指设置了 key name 的 tag，没有设置 key name 的 tagged 域在 json 化中使用的还是 filed key。

本段涉及的域处在同一层级，且该层级至少是一层嵌套层。

# 至少是这样的嵌套
type A struct {
    Name string
}

type B struct {
    Name string
}

type C struct {
    Name string
}

type D struct {
    A
    B
    C
}

func TestFeature(t *testing.T) {
    d := D{A{"A"}, B{"B"}, C{"C"}}
    r, _ := json.Marshal(d)
    fmt.Println(string(r))
}

以上 D struct 的 instance 有多个相同名称的域 Name。在对其做 marshal or unmarshal 时应用以下规则：

1、json tagged 的域优先于 untagged 的域考虑。

# 如果 A 的域被 `json:"Name"`，则 B C 里的 Name 域都被忽略。
{"Name":"A"}

2、如果 tagged 域的 tag key 唯一，则被正常 marshal or unmarshal，否则冲突的 tagged 域都被忽略。如果 untagged 域的 field key 唯一，则被正常 marshal or unmarshal，否则冲突的 untagged 域都被忽略。

# 如果 A 和 B 的 Name 域都被 `json:"Name"`，则视为  tag key 冲突，所以两者都被忽略。并且 C 的 Name 域为 untagged，所以 C instance 在这场“Name 域 json 化争夺战”中完全处于下风。最终这场战斗没有人胜出。
# {}

# 如果 A 的 Name 域被 `json:"Nickname"`，则剩下 B C 两个 untagged 域争夺，根据规则，最终没有在 json string 中见到 Name key。
# {"Nickname":"A"}

首先要明白什么是 anonymous struct field，看这里。

看过上面文章，应该对 anonymous struct field 及其规则有大致的了解。

在对 anonymous struct field 做 json encoding 时，anonymous struct field 中的 exported field 默认会被当做 outer struct 的 field 来 encoding。

如果对匿名结构域使用 json tag 功能给予 key name，则在 encoding 时不会被当作匿名结构域应用上述规则。

如果匿名结构域是 interface{} 类型，则也不会被当作匿名结构域应用上述规则，interface{} 的名称会被当作 key name。

举例：

type Kitchen struct {
    NumOfPlates uint
}

type Door interface {
    echo()
}

type FrontDoor struct {
    Name string
}

func (frontDoor *FrontDoor) echo() {
    fmt.Println("I'm " + frontDoor.Name)
}

type House struct {
    Kitchen    `json:"kitchen"`
    NumOfRooms uint
    Door
}

func main() {

    h := House{Kitchen{10}, 3, &FrontDoor{"Gold Door"}}

    sp, _ := json.Marshal(h)
    fmt.Println(string(sp))
}
# {"kitchen":{"NumOfPlates":10},"NumOfRooms":3,"Door":{"Name":"Gold Door"}}
# 正常被当作匿名结构域 encoding json 时：
# {"NumOfPlates":10,"NumOfRooms":3}

struct field 的 json encoding 行为可以通过 json tag 自定义，格式为：json:"keyName,option1,..."

以下是一些使用情况介绍：

1、即想使用 json tag 定义行为又要使用 struct field name 来作为 json key：json:",option1,..."

2、使用 omitempty 选项可以忽略该 field 的 json encoding，如果该 field 的值是空值的话。

3、使用 json:"-" 的 field 在 encoding 时被忽略。如果想要使用 "-" 做为 json key，那么在 "-" 后加上一个 comma 即可：json:"-,"。

4、如果你想要一些非 string 的类型被 encoding 为 json string，使用 json:",string" 标签选项即可。

type Person struct {
    Age uint `json:",string"`
}

func main() {
    p := Person{
        23,
    }
    sp, _ := json.Marshal(p)
    fmt.Println(string(sp))
}
# {"Age":"23"}

这里的 key name 不知道指什么，如果指的是 json tag 里给的 key name，那么在上面已经介绍过了，这里难道只再提一下作为 key name 的规则吗？

SciScore for 10.1101/2021.05.12.443228: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Table 2: Resources

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on pages 33, 37 and 38. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

Results from scite Reference Check: We found no unreliable references.

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

SciScore for 10.1101/2021.05.08.21256891: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Table 2: Resources

Results from OddPub: Thank you for sharing your data.

A potential limitation of our proof of concept method is that the use of intercalating dyes can make target-specific real-time detection challenging44, and could pose as a barrier for use in resource limited areas. Future lower resource implementations of our method could substitute EvaGreen for 100-200 µM halochromic dyes, like Cresol Red, in pH 8.8 buffered reaction mix to cause detectable colorimetric change when amplification products have been produced47. Alternatively more advanced detection methods utilizing sequence specific methods based on the use of probes (e.g. LAMP-BEAC23, OSD48, DARQ49 and QUASR50) could also be utilized as end-point detection methods, and remain to be tested in future work. Such methods would be advantageous as they could be multiplexed for different targets in the same reaction. QUASR, for instance, has been shown to allow single step and close-tube multiplexing50. The versions of both M-MLV and BstLF enzymes used in this paper are in the public domain in Chile but some of the M-MLV mutations are still subject to protection in other jurisdictions e.g. in the US (patent no. US7056716B251) with anticipated expiration date of 15 March 2021. Future work will, therefore, explore the use of off-patent enzymes Open-MMLV or HIV RT15 from the E. coli expression toolkit from ReClone initiative52 in order to have freedom-to-operate in any country. The use of public domain enzymes would also permit commercial partnerships seeking to scale-up production lo...

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: We did not find any issues relating to colormaps.

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

Results from scite Reference Check: We found no unreliable references.

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

SciScore for 10.1101/2021.05.11.443686: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Table 2: Resources

Results from OddPub: Thank you for sharing your data.

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: We did not find any issues relating to colormaps.

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

Results from scite Reference Check: We found no unreliable references.

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

SciScore for 10.1101/2021.05.10.443532: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Table 2: Resources

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on pages 56 and 54. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

• No conflict of interest statement was detected. If there are no conflicts, we encourage authors to explicit state so.
• No funding statement was detected.
• No protocol registration statement was detected.

Results from scite Reference Check: We found no unreliable references.

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

SciScore for 10.1101/2021.05.10.443524: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Table 2: Resources

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: We did not find any issues relating to colormaps.

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

Results from scite Reference Check: We found no unreliable references.

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

If you look at the incomplete graph.js implementation, you can see that it's actually using a class instead. This is good! However, I advocate for class names that happen to also be a hostname + resource (i.e., like URL, but omitting the http:// part).

(I suppose that, alternatively, if someone wanted to use a shorter class name, such as "w2g-tag", then graph.js could be configured to allow for that, but it would need to be explicit configuration on the author/publisher's part.)

SciScore for 10.1101/2021.05.09.442808: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Table 2: Resources

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on pages 34 and 31. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

Results from scite Reference Check: We found no unreliable references.

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

SciScore for 10.1101/2021.05.10.443404: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Table 2: Resources

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: We did not find any issues relating to colormaps.

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

Results from scite Reference Check: We found no unreliable references.

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

SciScore for 10.1101/2021.05.08.443267: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Table 2: Resources

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Limitations of study: Although a single intranasal administration of ChAd-SARS-CoV-2-S durably protected against SARS-CoV-2 variant replication in the upper and lower respiratory tract even ∼9 months after immunization, we note several limitations in our study. (1) We performed challenge studies in BALB/c mice transduced with hACE2 or C57BL/6 mice expressing an hACE2 transgene. Durability and protection studies will need to be corroborated in hamsters, non-human primates, and ultimately in humans. (2) Although our studies suggest that the mucosal immunity induced by intranasal vaccination could limit SARS-CoV-2 transmission, the use of mice precluded formal respiratory transmission analysis, which is better studied in hamsters and ferrets 46. (3) We observed robust protection in vivo against viruses displaying B.1.351 and B.1.1.28 spike proteins likely due to the high serum neutralizing antibody titers. Even though neutralizing antibody levels were lower with the variant strains due to mutations at sites in the receptor binding motif, the high starting level against the historical SARS-CoV-2 likely provided a sufficient cushion to overcome this loss in potency. Studies in other animals or with even lower doses of vaccine where neutralizing titers might be lower are needed to determine if the protective phenotype against variants of concern is maintained. (4) Finally, we did not establish the correlate of protection in these studies, as passive antibody transfer or T cell depl...

Results from TrialIdentifier: We found the following clinical trial numbers in your paper:<br><table><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Identifier</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Status</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Title</td></tr><tr><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NCT04751682</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Active, not recruiting</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Safety and Immunogenicity of an Intranasal SARS-CoV-2 Vaccin…</td></tr></table>

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: We did not find any issues relating to colormaps.

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

Results from scite Reference Check: We found no unreliable references.

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

SciScore for 10.1101/2021.05.04.441029: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Table 2: Resources

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on pages 20, 21 and 22. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

Results from scite Reference Check: We found no unreliable references.

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

SciScore for 10.1101/2021.05.10.443438: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Table 2: Resources

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: We did not find any issues relating to colormaps.

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

Results from scite Reference Check: We found no unreliable references.

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

SciScore for 10.1101/2021.05.09.443331: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Table 2: Resources

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Results from TrialIdentifier: We found the following clinical trial numbers in your paper:<br><table><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Identifier</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Status</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Title</td></tr><tr><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NCT03186781</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Completed</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Influenza HA Ferritin Vaccine, Alone or in Prime-Boost Regim…</td></tr><tr><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NCT03814720</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Completed</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Dose, Safety, Tolerability and Immunogenicity of an Influenz…</td></tr><tr><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NCT04579250</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Recruiting</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Dose, Safety, Tolerability and Immunogenicity of an Influenz…</td></tr><tr><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NCT04645147</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Recruiting</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Safety and Immunogenicity of an Epstein-Barr Virus (EBV) gp3…</td></tr><tr><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NCT04296279</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Recruiting</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">A Trial For The Study of Falciparum Malaria Protein 014 Admi…</td></tr><tr><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NCT04784767</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Recruiting</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">SARS-COV-2-Spike-Ferritin-Nanoparticle (SpFN) Vaccine With A…</td></tr></table>

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: We did not find any issues relating to colormaps.

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

Results from scite Reference Check: We found no unreliable references.

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

SciScore for 10.1101/2021.05.06.442911: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Table 2: Resources

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

While vaccines have been great successes, therapeutic biologics are emerging as a critical tool in preventing progression to severe disease in those who do become infected.(31) Several therapeutic antibody candidates with efficacy against SARS-2 have been recently identified, including three with emergency use authorization, but there are a number of caveats associated with conventional antibodies as therapeutics such as time-consuming discovery, relying on immunized or patient sera, expensive and labor-intensive production, and the large doses required to achieve clinical efficacy. (9, 22, 32-34) High stability, solubility, and the ability to be multimerized, are just some of the many reasons why single-domain heavy chain-based antibody therapeutics (VHH-Fc) represent a highly promising method for treatment.(12) One VHH-based therapeutic is already approved by the FDA for treatment of a rare blood clotting disorder and many more are in late stage clinical trials.(12, 35) The small size of VHH antibodies and the wider distribution of CDR3 loop lengths compared to human IgGs expands the type of epitope which can be effectively targeted.(12) Finally, preliminary evidence suggests that VHH-Fc antibodies are transported more efficiently into the blood and lung parenchyma following intraperitoneal administration compared to conventional IgG1 antibodies.(36, 37) Several groups have recently used in vitro screening techniques to identify VHH domains with high affinity for the SARS-2...

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

Results from JetFighter: We did not find any issues relating to colormaps.

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

Results from scite Reference Check: We found no unreliable references.

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

The brand name on the tag, ignoring the people who made these by hand

Is he talking about the name on the tag of the shirt?

less is more/better

SciScore for 10.1101/2021.05.04.21256571: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Table 2: Resources

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Results from TrialIdentifier: We found the following clinical trial numbers in your paper:<br><table><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Identifier</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Status</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">Title</td></tr><tr><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">ISRCTN51287266</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NA</td><td style="min-width:95px; border-right:1px solid lightgray; border-bottom:1px solid lightgray">NA</td></tr></table>

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: We did not find any issues relating to colormaps.

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
• No protocol registration statement was detected.

Results from scite Reference Check: We found no unreliable references.

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

SciScore for 10.1101/2021.05.04.21256472: (What is this?)

Please note, not all rigor criteria are appropriate for all manuscripts.

Table 1: Rigor

Table 2: Resources

Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

Results from TrialIdentifier: No clinical trial numbers were referenced.

Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

Results from JetFighter: We did not find any issues relating to colormaps.

• Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
• No funding statement was detected.
• No protocol registration statement was detected.

Results from scite Reference Check: We found no unreliable references.

About SciScore

SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

Author Response:

Reviewer #1:

In this manuscript the authors show that a designer exon containing a Fluorescent Protein insert can be used to edit vertebrate genes using an NHEJ based repair mechanism. The approach utilizes CRISPR to generate DSBs in intronic sequences of a target gene along with excision of a donor fragment from a co-transfected plasmid to initiate insertion of the exon cassette by ligation into the chromosome DSB.

I like the idea here of inserting FP sequences (and other tags) into introns in this way. Focusing on the N- and C-termini for insertions has always seemed arbitrary to me. In practice these internal sites may even tolerate tag insertions better than the termini. However, this remains to be seen.

My major reservation with this study is that the concepts here are not particularly novel. The approach is very similar to a concept already well established in gene-therapy circles of using introns as targets for inserting a super-exon preceded by a splice acceptor to correct inborn genetic lesions. The methodology employed is essentially HITI (https://www.nature.com/articles/nature20565).

What is new is the finding that FP insertions are frequently expressed and at least partly functional as evidenced by their ability to localize to the expected intracellular structures. However, no actual functional data is provided in this study so it remains to be seen how frequently the insertion of FP exons is tolerated. It would help the study substantilly to have functional information for a few insertions.

The value and utility of this study hinges on whether insertions of this type frequently retain function. The authors speculate that "labeling at an internal site of a gene is feasible as long as the insertion does not disrupt the function of the encoded protein. Many introns reside at the junctions of functional domains because introns have evolved in part to facilitate functional domain exchanges (Kaessmann et al., 2002; Patthy, 1999)." Thus an analysis of how often intron tags are tolerated as homozygotes would be helpful for users who will worry that a potentially "quick and dirty" CRISPIE insertion might not accurately report on the function and localization of their protein of interest.

We thank the reviewer for appreciating our idea. CRISPIE is indeed improved HITI, with the notable difference that the insertion takes place at the intronic region and that a designer intron/exon module is used. This design has a significant benefit in that INDELs in both labeled and unlabeled alleles will be unlikely to cause mutations at the levels of mRNA and proteins. CRISPIE is also different from the super-exon, which is now cited (Bednarski et al, 2016). CRISPIE does not involve the 3’ UTR and the poly A signal. This makes the donor template more standardized and smaller. Transcriptional controls embedded in endogenous introns after the editing sites can be retained in CRISPIE, but not when super-exons are used. We also achieve much higher efficiency in vivo than previous editing methods, which we feel is an important advance.

We now provide three different experiments to address the function of CRISPIEd β-actin and, in one experiment, the function of CRISPIEd α-tubulin 1B. One of the key functions of the cytoskeleton is to support growth. We now show that neither CRISPIE labeling of β-actin (hACTB), at two different intronic loci, and nor CRISPIE labeling of α-tubulin 1B (TUBA1B) affect the growth of U2OS cells (New Experiment #1; Figure 1H, and Figure 1-figure supplement 4), suggesting that labeled β-actin and α-tubulin are functional. In addition, as suggested, we now demonstrate that cells homozygous for CRISPIE insertions are viable and able to divide (New Experiment #2; Figure 4-figure supplement 1). We also show that two important neuronal functional parameters – the mEPSC frequency and amplitude – are not altered by CRISPIE labeling of hACTB in neurons in cultured hippocampal slices. (New Experiment #3; Figure 5– figure supplement 2).

Having shown the above results, we also hope to emphasize that, although CRISPIE provides a way to perform FP tagging of endogenous protein with high efficiency and low error rates, it cannot ensure that FP-tagging itself is benign for all proteins. Numerous studies have overexpressed FP-tagged proteins, which is well documented to have side effects. The CRISPIE method empowers researchers by allowing them to tag endogenous proteins without overexpression. However, if the FP-tagging itself affects protein function, CRISPIE will not be helpful. Each FP-tagging project, whether it is based on CRISPIE or other methods, will requires its own systematic characterization. We have now made this clear in the discussion (pg. 17): “… although CRISPIE enables the tagging of endogenous proteins with low error rates, it does not ensure that the tagged protein functions the same as the wild-type protein. Not all tagging is benign, and rigorous characterizations will be needed for each tagging experiment.”

Other comments:

1) Were homozygotes identified and were they viable in each instance?

We now provide data showing that cells homozygous for CRISPIE insertions are viable and able to divide (New Experiment #2; Figure 4-figure supplement 1).

2) You say: "The CRISPIE method should be broadly applicable for use with different FPs or with other functional domains, different protein targets, and different animal species." I don't know if you optimized your FP to avoid potential reverse strand splice acceptors, but some discussion of this important point should be made so that those trying to apply the approach will make sure that strong acceptors are not included accidentally in reverse oriented inserts.

Our RT-PCR does not detect reversed inserts at the mRNA level. We now add in the Discussion that donor design needs to eliminate unintended splicing sites in the reverse orientation. We write (pg. 17): “It should also be noted that, when designing the donor template, care should be given to not create unintended splicing acceptor sites in the inverted orientation. Otherwise, inverted insertion events can cause mutations at the mRNA and protein levels.”

3) Would your mRNA sequencing methodologies detect defective transcripts where the splice acceptor and a portion of the upstream FP exon was inserted causing a frame shifted and mispliced mRNA? Such mRNAs would be unstable due to NMD and thus not detected readily in a PCR based approach. Thus disruption of the mRNA by partial insertion of your donor (or fragments of the other co-injected DNA) might be much more widespread than is measured here. This could be tested by recovering clones that partially inserted the donor in the forward orientation and carefully monitoring for defects in mRNA splicing of the inserted allele. Were such clones detected and how frequently?

Our method should detect defective mRNAs, if they are not degraded. However, if defective mRNAs are quickly degraded, they are not measured in our current RT-PCR and NGS experiments, as described in Figure 2. While we cannot address this question directly, we now provide evidence that the cell growth and neuronal function after CRISPIE labeling of β-actin remain normal.

We also thank the reviewer for suggesting the cloning approach. This proposed experiment, however, may potentially be affected if potential defective mRNAs can result in decreased cell survival/growth. Although this experiment will require time beyond the three-month revision period expected by eLife due to the length of time required to clone cells, we will keep this in mind in our future efforts.

4) You note that in the case of vinculin the coding sequence of the last exon of hVCL was included in the insertion donor sequence, and a stop codon was introduced at the end of the mEGFP coding sequence. This is essentially the strategy for super-exon insertion into targets for gene therapy, instead of a splice donor on the C-terminus you include a stop codon. You should site these previous studies. Inclusion of a stop codon in frame would be expected to cause NMD, did you also include transcription termination signals?

NMD will happen if the stop codon is further than about approximately 50 nucleotides upstream of any exon-junction complexes (Lewis et al, PNAS 2009). However, NMD won’t occur if it is within 50 nucleotides. For example, synaptophysin – a highly expressed neuronal protein – has its stop codon at its second to last exon within 50 nucleotides of the exon junction. The stop codon we used for labeling hVCL is also within 50 nucleotides (~20 nt) of the exon junction.

We now cite Bernarski et al, 2016, which describes the use of super-exons in gene therapy. At the same time, we think that our approach is still different from the super-exon concept. After the stop codon, the 3’ UTR is not included. Instead, a splicing donor is included, allowing the exon to be spliced to the subsequent endogenous exon. This allows the insert to remain small for high insertion efficiency and makes it easy to produce the template (some 3’ UTRs can be several kilobase pairs in length), while utilizing the endogenous translational controls built into the native 3’ UTR.

Reviewer #2:

In-frame insertion of fluorescent protein tags into endogenous genes allows observation of protein localization at native expression levels, and is therefore an essential approach for quantitative cell biology. Once limited to unicellular model organisms such as yeast, endogenous gene tagging has become well-established in invertebrate model systems such as C. elegans and Drosophila since the advent of CRISPR technology in the last decade. However, a robust and widely accepted endogenous gene tagging strategy for mammalian cells has remained elusive. This is largely due to the fact that homologous recombination, the method used to create knock-ins in invertebrates, is inefficient (or sometimes doesn't work at all) in mammalian cells, especially those that do not divide rapidly.

Several studies have attempted to bypass the need for homologous recombination by using a different method, non-homologous end joining (NHEJ) to insert GFP tags into vertebrate genomes (e.g. Auer et al. Genome Res 2014; Suzuki et al. Nature 2016; Artegiani et al. Nature Cell Biol. 2020). Such approaches can be orders of magnitude more efficient than homologous recombination, but the generated alleles require careful validation because of the error-prone nature of NHEJ.

Here, Zhong and colleagues improve upon the existing NHEJ-based gene tagging approaches by designing synthetic exons (comprising a FP coding sequence with 5' and 3' splice sites) that can be inserted into native introns using NHEJ. The beauty of this approach is that any mutations (indels) created by the error-prone NHEJ repair mechanism are spliced out, and therefore do not affect the sequence of the encoded protein. A limitation is that tags must be inserted internally within a protein of interest and cannot be targeted to the extreme N- or C-terminus, but this limitation is clearly stated and discussed by the authors. Overall, this is a novel (to my knowledge) and powerful strategy that is likely to advance the field.

We thank the reviewer for the very positive comments regarding our CRISPIE method.

Reviewer #1 (Public Review):

In this manuscript the authors show that a designer exon containing a Fluorescent Protein insert can be used to edit vertebrate genes using an NHEJ based repair mechanism. The approach utilizes CRISPR to generate DSBs in intronic sequences of a target gene along with excision of a donor fragment from a co-transfected plasmid to initiate insertion of the exon cassette by ligation into the chromosome DSB.

I like the idea here of inserting FP sequences (and other tags) into introns in this way. Focusing on the N- and C-termini for insertions has always seemed arbitrary to me. In practice these internal sites may even tolerate tag insertions better than the termini. However, this remains to be seen.

My major reservation with this study is that the concepts here are not particularly novel. The approach is very similar to a concept already well established in gene-therapy circles of using introns as targets for inserting a super-exon preceded by a splice acceptor to correct inborn genetic lesions. The methodology employed is essentially HITI (https://www.nature.com/articles/nature20565).

What is new is the finding that FP insertions are frequently expressed and at least partly functional as evidenced by their ability to localize to the expected intracellular structures. However, no actual functional data is provided in this study so it remains to be seen how frequently the insertion of FP exons is tolerated. It would help the study substantilly to have functional information for a few insertions.

The value and utility of this study hinges on whether insertions of this type frequently retain function. The authors speculate that "labeling at an internal site of a gene is feasible as long as the insertion does not disrupt the function of the encoded protein. Many introns reside at the junctions of functional domains because introns have evolved in part to facilitate functional domain exchanges (Kaessmann et al., 2002; Patthy, 1999)." Thus an analysis of how often intron tags are tolerated as homozygotes would be helpful for users who will worry that a potentially "quick and dirty" CRISPIE insertion might not accurately report on the function and localization of their protein of interest.

Other comments:

1) Were homozygotes identified and were they viable in each instance?

2) You say: "The CRISPIE method should be broadly applicable for use with different FPs or with other functional domains, different protein targets, and different animal species." I don't know if you optimized your FP to avoid potential reverse strand splice acceptors, but some discussion of this important point should be made so that those trying to apply the approach will make sure that strong acceptors are not included accidentally in reverse oriented inserts.

3) Would your mRNA sequencing methodologies detect defective transcripts where the splice acceptor and a portion of the upstream FP exon was inserted causing a frame shifted and mispliced mRNA? Such mRNAs would be unstable due to NMD and thus not detected readily in a PCR based approach. Thus disruption of the mRNA by partial insertion of your donor (or fragments of the other co-injected DNA) might be much more widespread than is measured here. This could be tested by recovering clones that partially inserted the donor in the forward orientation and carefully monitoring for defects in mRNA splicing of the inserted allele. Were such clones detected and how frequently?

4) You note that in the case of vinculin the coding sequence of the last exon of hVCL was included in the insertion donor sequence, and a stop codon was introduced at the end of the mEGFP coding sequence. This is essentially the strategy for super-exon insertion into targets for gene therapy, instead of a splice donor on the C-terminus you include a stop codon. You should site these previous studies. Inclusion of a stop codon in frame would be expected to cause NMD, did you also include transcription termination signals?

DOI: 10.1016/j.stem.2020.11.019

Resource: (IMSR Cat# CRL_022,RRID:IMSR_CRL:022)

Curator: @scibot

SciCrunch record: RRID:IMSR_CRL:022

What is this?