Establishing a Hoxb8 cell model of Severe Congenital Neutropenia

Severe congenital neutropenia (SCN) is a rare genetic condition characterized by low neutrophil counts, severe bacterial infections, and cancer predisposition. Many genetic variants found in patients with SCN are in genes associated with mitochondrial function and homeostasis; their mutation causes differentiation arrest at the granulocyte-monocyte progenitor (GMP) cell stage. HoxB8 cells are murine derived bone marrow progenitor cells immortalized at the GMP stage. We strive to generate a HoxB8 model of SCN so we can study this rare precursor population and provide a system for additional genetic manipulation of these cells. We generated HoxB8 cells from a Rosa26 Cas9 knock-in mouse and transduced these cells with guide RNA targeted to known SCN genes ELANE and CLPB. By using CRISPR editing, sequencing, and a tracking indels by decomposition (TIDE) assay, we investigated the relationship between ELANE and CLPB genes and HoxB8 differentiation. We found that ELANE edited and CLPB edited cells with predicted knockout variants were underrepresented in the population that differentiated into neutrophils. These data support the future use of our Cas9 HoxB8 Cells to identify genes critical for neutrophil differentiation in a CRISPR screen.