Cas9 + conditionally immortalized neutrophil progenitors as a tool for genome wide CRISPR screening for neutrophil differentiation and function
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eLife Assessment
In this manuscript, Jong et al. provide and validate a very useful resource for performing CRISPR screenings to study neutrophil differentiation and function by generating Hoxb8 cells that constitutively express Cas9. This library-screening approach has the potential to improve on the established lentiviral CRISPR-Cas9 editing of Hoxb8 cells. However, the technical advances provided are only incremental and the results presented in this study do not significantly further our understanding of these cells, but rather provide a good validation of their Cas9+ modified version.
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Abstract
Neutrophils are short-lived cells of the innate immune system that play numerous roles in defense against infection, regulation of immune responses, tissue damage and repair, autoimmunity, and other non-communicable diseases. Understanding neutrophil function at a mechanistic level has been hampered by the difficulty of working with primary neutrophils, which die rapidly upon isolation, and the relative paucity of neutrophil cell lines. Murine neutrophil progenitors that are immortalized with estrogen-regulated expression of Hoxb8 differentiate into neutrophils upon withdrawal of estrogen and facilitate the quantitative production of neutrophils in vitro . Here we report the creation of a Cas9 + ER-Hoxb8 neutrophil progenitor cell line that enables both forward and reverse genetic analysis of neutrophils for the first time. By editing progenitors via transduction with sgRNAs, and then withdrawing estrogen, Cas9 edited neutrophils are produced with high efficiency. Importantly, neutrophil differentiation of edited progenitors occurs both in vitro in cell culture and when transferred into murine recipients. To demonstrate the utility of Cas9 + ER-Hoxb8 progenitors for forward genetics, we performed a pooled CRISPR screen to identify factors required for survival during neutrophil differentiation. This screen identified hundreds of genes both negatively and positively selected under differentiation conditions. One of the top hits from this screen was Cebpe , a transcription factor known to be required for neutrophil differentiation from pre-neutrophils to immature neutrophils. Using the progenitor cell line, we also confirmed that Cepbe is required for neutrophil differentiation in vivo , validating the utility of this cell line both for screening and for studying in vivo phenotypes. The genome-wide screen also identified all components of the WASH complex as being required for neutrophil differentiation, a finding that extends the known role of WASH in hematopoietic stem cell differentiation to later stages of neutrophil development. Taken together, we demonstrate that Cas9 + ER-Hoxb8 immortalized neutrophils can be used to study neutrophil function both in vitro and in vivo . This new resource will enable the analysis of the role of neutrophils in numerous disease states using genetics for the first time.
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eLife Assessment
In this manuscript, Jong et al. provide and validate a very useful resource for performing CRISPR screenings to study neutrophil differentiation and function by generating Hoxb8 cells that constitutively express Cas9. This library-screening approach has the potential to improve on the established lentiviral CRISPR-Cas9 editing of Hoxb8 cells. However, the technical advances provided are only incremental and the results presented in this study do not significantly further our understanding of these cells, but rather provide a good validation of their Cas9+ modified version.
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Reviewer #1 (Public Review):
This study aims to develop a new system to analyze genetic determinants of neutrophil function by large-scale genetic screens. For that, the authors use a genetically-engineered ER-Hoxb8 neutrophil progenitor line that expresses Cas9 to perform CRISPR screens and to identify genes required for neutrophil survival and differentiation.
A main strength of this study is that the authors develop a pooled CRISPR sgRNA library applicable to neutrophils and show potential determinants of neutrophil differentiation in vitro using this screening methodology.
A main weakness of this study is that identified candidates associated with neutrophil differentiation, as those indicated in Fig. 4A, were not validated in vivo using neutrophil-specific K.O. models or further characterized in vitro (e.g. transcriptional or …
Reviewer #1 (Public Review):
This study aims to develop a new system to analyze genetic determinants of neutrophil function by large-scale genetic screens. For that, the authors use a genetically-engineered ER-Hoxb8 neutrophil progenitor line that expresses Cas9 to perform CRISPR screens and to identify genes required for neutrophil survival and differentiation.
A main strength of this study is that the authors develop a pooled CRISPR sgRNA library applicable to neutrophils and show potential determinants of neutrophil differentiation in vitro using this screening methodology.
A main weakness of this study is that identified candidates associated with neutrophil differentiation, as those indicated in Fig. 4A, were not validated in vivo using neutrophil-specific K.O. models or further characterized in vitro (e.g. transcriptional or epigenetic changes during maturation when compared to non-targeting sgRNA controls).
As secondary strengths, the authors provide evidence of efficient gene editing in Cas9+ER-Hoxb8 neutrophils both in vivo and in vitro and provide evidence of the specificity of this methodology using a Cas9+ER-Hoxb8 immortalized cell line that differentiates into macrophages.
In terms of methodology, this study provides a useful tool to explore gene regulatory networks in neutrophils in large-scale genetic screens. However, it falls short in identifying and validating the true potential of this kind of methodology in the biology of the neutrophil.
Moreover, the technical advances in the field are only incremental as several studies, including those using CRISPR/Cas9 technology in Hoxb-8 immortalized neutrophil progenitor cell lines have been already performed.
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Reviewer #2 (Public Review):
In this manuscript, Jong et al. provide and validate a very useful resource for performing CRISPR screenings to study neutrophil differentiation and function. The major strength of the paper lies in its careful validation of many aspects of the Hoxb8-immortalized progenitor cells, including their differentiation capacity, their ability to clear bacteria, and their capacity to differentiate in vivo. The authors succeed at this, with results correctly supporting their conclusions. The major weaknesses are its presentation and writing, some of which are poorly organized. Finally, while the potential impact of this resource in the field could be very large, the CRISPR screening results appear half-baked, almost preliminary, and could be better validated, or at least presented. A few other points that warrant …
Reviewer #2 (Public Review):
In this manuscript, Jong et al. provide and validate a very useful resource for performing CRISPR screenings to study neutrophil differentiation and function. The major strength of the paper lies in its careful validation of many aspects of the Hoxb8-immortalized progenitor cells, including their differentiation capacity, their ability to clear bacteria, and their capacity to differentiate in vivo. The authors succeed at this, with results correctly supporting their conclusions. The major weaknesses are its presentation and writing, some of which are poorly organized. Finally, while the potential impact of this resource in the field could be very large, the CRISPR screening results appear half-baked, almost preliminary, and could be better validated, or at least presented. A few other points that warrant revision are included below:
The introduction should be better constructed and organized. It should be written with more connectors to present facts in a stream that flows naturally, from the broad general facts to the experimental details implemented in the manuscript. It should also discuss other similar approaches used in the literature, such as LaFleur et al. 2019, and relate in which ways these presented methods could be better.
The scheme in Figure 4A should more clearly indicate the timings, doublings, numbers of cells, and other aspects of the experimental design.
The volcano plot in Figure 4B is poorly informative and almost redundant. What does one make of it?
The representation (normalized reads) of each sgRNA in the library and across multiple experiments, including their correlation, should be checked and plotted, to visualize how robust these replicates are.
In Figure 4E, the distribution of the hit sgRNAs should be compared to all other targeting guides (instead of just to non-targeting controls). Linear density distribution plots or scatter plots of all guides are usually the best way, but there are others (for example, see Figure 4 of LaFleur et al. 2019). Ideally, each independent sgRNA for each gene in the library, as well as biological replicates, should be separately shown, with hits clearly highlighted.
While in vivo differentiation is shown as possible with these cell lines, it is unclear whether CRISPR screenings could be performed in vivo too. Would sgRNA representation suffice for genome-wide? At least some of the new hits could be validated by testing differentiation in vivo (i.e. WASH complex).
In the methods section, the RNA-seq analysis pipeline details are missing (versions, software for alignment, quantification, differential gene expression, and enrichment). Also, parameters for MAGeCK and MAGeCKFlute should be explicit and detailed.
The discussion is mostly a summary of the results. It is lacking in detail and thoughtful discussion regarding novelty and impact beyond the validation of the cell line. What about potential applications? What about extending screenings to test bacterial-killing, as suggested in Figure 2? What about limitations compared to other similar methods out there? There is little discussion of such important potential matters. Also, a large part of the discussion is dedicated to discussing details about Cebpe that are all well known in the literature and add little value.
Figure legends are typically too succinct and hard to interpret, especially for non-experts. The text should enable the figure reader to correctly interpret what is shown in each panel.
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Reviewer #3 (Public Review):
Primary neutrophils are difficult to modify genetically, whereas the generation of knockout mice to study the role of specific proteins is time-consuming and expensive. CRISPR-Cas 9 genetic modification of neutrophil progenitors in vitro offers a platform to study neutrophil biology. Hoxb8 cells are immortalized neutrophil progenitors that differentiate into neutrophils when cultured in the presence of G-CSF, and have been shown to recapitulate the stages of murine neutrophil differentiation. They have also been shown to be amendable to CRISPR-Cas 9 genetic editing with successful deletion of key transcriptional regulators of neutrophil maturation and function. The authors of this manuscript offer an extension to this technique, by generating Hoxb8 cells that constitutively express Cas9. This may reduce the …
Reviewer #3 (Public Review):
Primary neutrophils are difficult to modify genetically, whereas the generation of knockout mice to study the role of specific proteins is time-consuming and expensive. CRISPR-Cas 9 genetic modification of neutrophil progenitors in vitro offers a platform to study neutrophil biology. Hoxb8 cells are immortalized neutrophil progenitors that differentiate into neutrophils when cultured in the presence of G-CSF, and have been shown to recapitulate the stages of murine neutrophil differentiation. They have also been shown to be amendable to CRISPR-Cas 9 genetic editing with successful deletion of key transcriptional regulators of neutrophil maturation and function. The authors of this manuscript offer an extension to this technique, by generating Hoxb8 cells that constitutively express Cas9. This may reduce the variation between the generated knock-out cells by avoiding the introduction of Cas9 in a plasmid every time together with a guide RNA.
The first part of the manuscript is dedicated to the characterisation of Cas9+HoxB8 cells throughout their differentiation. Considering the existing body of literature on HoxB8 progenitors and their differentiation into neutrophils ex vivo, it does not significantly further our understanding of these cells, but rather provides a good validation to their Cas9+ modified version of them. Gene editing using Cas9+ Hoxb8 progenitors seems to be highly efficient, which is an important technical point, however, it is hard to assess the degree of improvement in efficiency compared to the published protocols with Cas9 delivery in a plasmid.
As a test, the authors use Cas9+HoxB8 progenitor to generate a knockout of CEBPE, known for its important role in neutrophil development. They convincingly demonstrate its impact on HoxB8 cell differentiation, with in vivo reconstitution of wild-type and CEBPE-deficient HoxB8 progenitors into irradiated mice being especially elegant. However, the transfer into different recipient mice assumed no differences in the recipient environment, while immunophenotyping for mature neutrophils within the HoxB8 progenitor-derived cells did not account for possible differences in numbers of wt and CEBPE KO surviving cells, limiting the conclusions.
Finally, the authors put the system to the test by screening a library of Brie gRNA library of ~80K mouse sgRNAs, targeting almost 20K genes with 4 gRNA per gene coverage, to identify genes that are needed for the differentiation of Cas9+ERHoxb8 progenitors into mature neutrophils. They identify a number of hits, amongst which the WASH complex and CEBPE are highlighted. A comparison of cell numbers prior to differentiation and at 4 days post differentiation indicates that they are indeed required for neutrophil survival. To validate the role of these hits in neutrophil maturation itself, as they stated in the aims, i.e. "to identify genes that modulate the differentiation of Cas9+ERHoxb8 progenitors into mature neutrophils", phenotypic, functional, and morphological characterization of these cell lines could have been appropriate.
Overall, this study has the potential to improve on the established lentiviral CRISPR-Cas9 editing of Hoxb8 cells and be valuable for library-screening approaches for neutrophil modulators, which will benefit the community.
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