PPATHOGENS-D-25-01952

CRISPRi-seq in Haemophilus influenzae reveals genome-wide and medium-specific growth determinants

PLOS Pathogens

Dear Dr. Garmendia,

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D. Ashley Robinson, Ph.D.

Academic Editor

PLOS Pathogens

D. Scott Samuels

Section Editor

PLOS Pathogens

Sumita Bhaduri-McIntosh

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0003-2946-9497

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

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Reviewers' Comments:

Reviewer's Responses to Questions

Part I - Summary

Please use this section to discuss strengths/weaknesses of study, novelty/significance, general execution and scholarship.

Reviewer #1: This paper presents a fascinating development in functional genomics to define essential genes and explore fitness impacts across the entire H. influenza genome. There are current limitations (most notably use of model strain Rd which is not fully representative of either encapsulated or nonencapsulated lineages in carriage or disease), and the conditions for which fitness is tested are fairly limited...reviewer would certainly like to see this work extended into more relevant strains/lineages and conditions associated with carriage or virulence. These points certainly don't limit the significance or impact of the work in this paper

Reviewer #2: This manuscript by Gil-Campillo et al. describes the implementation and validation of an inducible CRISPR system for mutant generation in Haemophilus influenzae. The manuscript is well written and presented in a clear, logical manner that is well supported by the data. The approach is sound and strengthened by the use of varied methodology and phenotypic complementation. While the CRISPRi approach has been described in other bacteria, there is novelty in its application to Haemophilus as well as in the development of the HaemoBrowse database generated in this work. The validation of identified genes through direct mutagenesis, complementation, and phenotypic assays is a significant strength of the study.

Reviewer #3: PloS review Sept.23, 2025

This is a commendable study in which the authors report a comprehensive functional genomics tool for H. influenzae strain Rd KW20. While primarily a methods paper, the authors demonstrate cell and physiological phenotypes, such as cell division and nutrient utilization, that can be studied using the sgRNA conditional expression system. Overall, the findings are straightforward and confirm or extend results of previous essential gene identifications provided by transposon insertion site sequencing studies and demonstrate utility of an inducible genetic repression system that allows detailed confirmation and follow-up functional analyses of genes involved in fitness under varied conditions.

It would be useful to know the approximate percentage of sgRNA constructs that are functionally able to deplete their target RNAs.

From the standpoint of studying H. influenzae as a pathogen, a missed opportunity was the lack of an animal infection study, as the tool could potentially clarify which of the in vitro essential genes are also essential for infection.

Line 180: The term 'genetic features' is vague. Describe the type of elements that were targeted by the design. (eg. all protein coding genes, RNA genes, +?).

Lines 319-322: Some studies conducting transposon-based essential gene identification in H. influenzae were omitted and could improve the comparision. The comparison of the identified essential gene set with those generated in transposon studies should be reported directly as a list, with some discussion of genes that were discordant between methods. Also, were there any genes identified that were not found in transposon-based studies?

Lines 337-338: This sentence confounds an experimental caveat (mixed competition) with statistical significance.

Line 348: The term "showcased medium specificities" is ambiguous. Do the authors mean differences in media composition? If so, this is circular logic as we already know the media compositions are different, and the main constituents are known, so it is not clear why it would need to be showcased.

Lines 355-359: The phrase "differentially essential gene enrichment in sBHI" should be clearly defined. The specific genes under discussion should be named and citations provided for the functions ascribed to these genes. The data supporting this discussion should be included in the manuscript so that readers do not need to access the online portal to interpret the statements.

Line 356: The authors should comment on whether the insertion of all of the sgRNA constructs into the xylose locus may have influenced essentiality results seen with xylose transport genes.

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Part II – Major Issues: Key Experiments Required for Acceptance

Please use this section to detail the key new experiments or modifications of existing experiments that should be absolutely required to validate study conclusions.

Generally, there should be no more than 3 such required experiments or major modifications for a "Major Revision" recommendation. If more than 3 experiments are necessary to validate the study conclusions, then you are encouraged to recommend "Reject".

Reviewer #1: As noted, ideally this would be extended to relevant strains or conditions, including in vitro or in vivo infection models. I think it's fair for that to be mentioned in discussion as an important future goal

Reviewer #2: None

Reviewer #3: (No Response)

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Part III – Minor Issues: Editorial and Data Presentation Modifications

Please use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity.

Reviewer #1: No Concerns

Reviewer #2: For many of the induced mutants, outgrowth is observed around 8–10 hours under several assay/growth conditions. This observation should be more directly addressed in the Discussion. Is this due to loss of induction or the emergence of suppressor mutants? Some discussion or a working hypothesis should be provided.

Throughout, “MI-V” should be changed to “M-IV” for consistency with the original cited publication.

Line 296: remove the repeated word “designed”

Reviewer #3: (No Response)

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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Figure resubmission:

Reproducibility:

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Dear Dr. Garmendia,

We are pleased to inform you that your manuscript 'CRISPRi-seq in Haemophilus influenzae reveals genome-wide and medium-specific growth determinants' has been provisionally accepted for publication in PLOS Pathogens.

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Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Pathogens.

Best regards,

D. Ashley Robinson, Ph.D.

Academic Editor

PLOS Pathogens

D. Scott Samuels

Section Editor

PLOS Pathogens

Sumita Bhaduri-McIntosh

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0003-2946-9497

Michael Malim

Editor-in-Chief

PLOS Pathogens

orcid.org/0000-0002-7699-2064

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Reviewer Comments (if any, and for reference):