Cas9+ Conditionally Immortalized Neutrophil Progenitors as a Tool for Genome Wide CRISPR Screening for Neutrophil Differentiation and Function

Why It Matters

Because primary neutrophils are short‑lived and hard to manipulate, this immortalized, CRISPR‑compatible line provides a scalable tool for dissecting neutrophil development and function, accelerating therapeutic target discovery for infectious, inflammatory and autoimmune disorders.

Key Takeaways

• Cas9+ER‑Hoxb8 line produces neutrophils after estrogen withdrawal
• CRISPR screen identified Cebpe and WASH complex as essential genes
• Edited neutrophils differentiate both in culture and in mouse recipients
• Platform enables forward and reverse genetics for neutrophil research
• Data deposited in GEO (GSE211699) for community reuse

Pulse Analysis

Neutrophils are the most abundant white‑blood cells and a first line of defense against pathogens, yet their study has been hampered by rapid cell death after isolation and a scarcity of robust cell lines. Traditional primary‑cell approaches limit high‑throughput experiments, creating a bottleneck for researchers seeking to map the genetic circuitry that governs neutrophil development, activation, and disease‑related dysfunction. The new Cas9+ER‑Hoxb8 progenitor line directly addresses these challenges by providing a renewable, manipulable source of neutrophils that retain physiological relevance.

The engineered cell line combines three key innovations: constitutive Cas9 expression for seamless CRISPR editing, estrogen‑regulated Hoxb8 to maintain progenitor proliferation, and a conditional differentiation step triggered by estrogen withdrawal. Researchers can introduce sgRNA libraries into progenitors, expand the edited pool, then induce maturation into neutrophils that exhibit expected morphology and functional markers. Validation experiments confirmed that edited cells differentiate efficiently both in culture dishes and after engraftment into murine hosts, offering a rare in vivo testing platform for genetic hits.

By leveraging this system, the authors performed a genome‑wide pooled CRISPR screen that highlighted hundreds of genes essential for neutrophil survival, notably confirming the known role of Cebpe and uncovering the WASH complex as a novel regulator of later‑stage differentiation. The availability of the RNA‑seq and screening datasets (GEO GSE211699) invites the broader community to explore neutrophil biology across infection, autoimmunity, and cancer contexts. As a scalable, genetically tractable model, the Cas9+ER‑Hoxb8 line is poised to accelerate target validation, drug screening, and mechanistic studies that were previously infeasible with primary neutrophils.