CLPB-deficient HSPCs have impaired granulocytic differentiation. Human cord blood CD34⁺ cells were nucleofected with guide RNA targeting CLPB (CLPB-g1 or CLPB-g3) or a control guide RNA targeting the intron of AAVS1 (as a gene editing control) complexed together with recombinant Cas9 protein. Edited CD34⁺ cells were cultured for 7-14 days in media containing G-CSF and SCF. (A) Representative flow plots showing gating strategy to identify CD11b⁺CD16⁺ mature neutrophils (PMN) or CD11b^+/−CD16⁻ granulocytic precursors; data are gated on CD14⁻ cells to remove monocytes. Data are quantified in the right panel. (B) Representative hematoxylin/eosin-stained cytospin preparations of cells on day 14 of culture; original magnification using a 63X objective. Data are quantified in the right panel. (C) Number of CFU-G per 2000 gene-edited CD34⁺ cells. (D) Fold change (from day 0) in edited cells (for CLPB, edited out-of-frame) or unedited cells (for CLPB, unedited plus edited in-frame) in PMNs or granulocytic precursors sorted on day 14. (E) Percentage of caspase-3⁺ granulocytic precursors on day 7 of culture. (F) Cells were cultured for 7 days, and cell cycle was assessed by flow cytometry. Data represent 3-5 independent experiments. *P < .05, **P < .01, ***P < .005, repeated measures 1-way ANOVA.