Cas9+ conditionally immortalized neutrophil progenitors as a tool for genome wide CRISPR screening for neutrophil differentiation and function
- University of California, Berkeley, United States
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. Here, we report the creation of a Cas9+ER-Hoxb8 neutrophil progenitor cell line that enables both forward and reverse genetic analysis of neutrophils. 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, including Cebpe, a transcription factor known to be required for neutrophil differentiation from pre-neutrophils to immature neutrophils. Using this progenitor cell line, we confirmed that Cebpe is required for neutrophil differentiation in vivo, validating the utility of this line for studying in vivo phenotypes. The screen also identified all components of the WASH complex as being required for neutrophil differentiation, extending its known role in hematopoietic stem cell differentiation to later stages of neutrophil development. Taken together, this resource enables the analysis of the role of neutrophils in numerous disease states using genetics for the first time.
Data availability
RNAseq data is uploaded into GEO: GSE211699Processed RNAseq data has been included as a supplemental attachment.CRISPR screening data is included as a supplemental attachment.All data generated or analyses during this study are included in the manuscript and supporting file.
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Transcriptional profile of Cas9+HoxB8 neutrophil progenitors at day 0 and day 2 after withdrawal of estrogenNCBI Gene Expression Omnibus, GSE211699.
Article and author information
Author details
Funding
NIH Office of the Director (U19AI135990-01)
- Sarah A Stanley
National Science Foundation Graduate Research Fellowship Program (DGE-1752814)
- Robyn M Jong
NIH Office of the Director (T32 GM 7232-40)
- Robyn M Jong
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: All procedures involving the use of mice were approved by the University of California, Berkeley Institutional Animal Care and Use Committee (protocol 2015-09-7979). All protocols conform to federal regulations, the National Research Council Guide for the Care and Use of Laboratory Animals, and the Public Health Service Policy on Humane Care and Use of Laboratory Animals.
Copyright
© 2026, Jong et al.
This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.
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Cas9+ conditionally immortalized neutrophil progenitors as a tool for genome wide CRISPR screening for neutrophil differentiation and function
eLife 15:e82289.
https://doi.org/10.7554/eLife.82289
