Cas9+ conditionally immortalized neutrophil progenitors as a tool for genome-wide CRISPR screening for neutrophil differentiation and function
- Department of Molecular and Cell Biology, Division of Immunology and Pathogenesis, University of California, Berkeley, United States
- School of Public Health, Division of Infectious Diseases and Vaccinology, University of California, Berkeley, United States
Figures
Figure 1
Differentiated Cas9+ER-Hoxb8 differentiated neutrophils closely resemble primary neutrophils.
(A) Cas9+ER-Hoxb8 cells differentiated for 0, 3, and 4 days were analyzed for expression of indicated myeloid markers by flow cytometry. (B) Flow cytometry analysis of CD11b and Ly6G staining after 4 days of differentiation in the presence and absence of G-CSF. (C, D) Transmission electron microscopy (TEM) of Cas9+ER-Hoxb8 before (C) differentiation (D) and after 4 days of differentiation. (E) Comparison of gene expression profile of Cas9+ER-Hoxb8 after 2 days of differentiation with RNAseq data from the Immunology Genomes database. Y-axis represents log2(gene expression value/average expression value of all genes) for different cell types; in order from left to right: macrophages (MF_PC, MF_Fem_PC, MF_226+II + 480lo_PC, MF_RP_Sp, MF_Alv_Lu, MF_pIC_Alc_Lu, MF_microglia_CNS, MF_AT), monocytes (Mo_6C+II-_Bl, Mo_6C-II-), neutrophils (GN_BM, GN_Sp, GN_Thio_PC), and mast cells (MC_heparainase_PC). All data are representative of at least two experiments.
Figure 2
Differentiated Cas9+ER-Hoxb8 progenitors recapitulate primary neutrophil responses to methicillin resistant Staphylococcus aureus (MRSA) and M. tuberculosis.
(A) Differentiated neutrophils were infected at the indicated multiplicities of infection (MOI) with MRSA USA300 and bacterial number (CFU) was measured immediately after phagocytosis. (B) Differentiated neutrophils effectively kill MRSA over a timecourse of infection as measured by CFU at indicated timepoints. (C) Differentiated neutrophil survival assayed using CellTiter Glo after infection with M. tuberculosis Erdman strain. Bacterial numbers were assayed using a luciferase (lux) reporter. All experiments were repeated a minimum of three times.
Figure 3 with 1 supplement
Efficient CRISPR/Cas9 editing in differentiated Cas9+ER-Hoxb8 cells in vitro and in vivo.
(A) Editing efficiency of individual single guide RNAs (sgRNAs) was analyzed using TIDE analysis. (B) Edited Cas9+ ER-Hoxb8 progenitor cells were transferred into irradiated mice, and differentiation was analyzed at 5 days post transfer. (C, D, E) Flow cytometry analysis of in vivo differentiation of neutrophil progenitors at 5 days post transfer into irradiated recipients using progenitors edited with a control guide (C, E) or with a sgRNA targeting Cebpe (D, E). Neutrophils are identified as Ly6Ghi and CD11b positive. All experiments were repeated 2–3 times. Unpaired t-test p-value calculated in comparison with nontargeting control cell line. ****p<0.0001.
Figure 3—figure supplement 1
Time course of differentiation of Hoxb8+ neutrophil progenitors in vivo.
Percentage of cells was measured using flow cytometry for indicated markers. Experiment was repeated twice with one mouse per cell line.
Figure 4 with 1 supplement
CRISPR screen using Cas9+ER-Hoxb8 library reveals factors required for survival during neutrophil, but not macrophage, differentiation.
(A) Schematic of the CRISPR screen. Cas9+HoxB8 library was created. Screening is initiated by growing up cells for a minimum of 500 x coverage. These cells are differentiated by estradiol withdrawal for 4 days, at which time DNA is prepared and sequenced (B) Volcano plots of results from the CRISPR screen. (C) GSEA using MAGeCK Flute analysis depicts GO cellular compartment categories negatively enriched in the differentiation screen. (D) Members of the WASH complex. (E, F) Survival of individual Cas9+ER-Hoxb8 progenitor cell lines targeted with individual single guide RNA (sgRNA) selected from the Brie library was determined prior to differentiation and at 4 days post-differentiation. The same sgRNAs were used to target both neutrophil (E) and macrophage (F) progenitors. All experiments were repeated a minimum of two times. The CRISPR screen was performed in biological triplicate. *p<0.05, **p<0.01, ***p<0.001 by one-way ANOVA with multiple comparisons.
Figure 4—figure supplement 1
CRISPR pooled screen single guide RNA (sgRNA) read counts.
(A) Read counts of control sgRNA (red) and targeting sgRNA (blue) from the Brie library of an undifferentiated replicate. (B) Distribution of the fraction of unique sgRNA having a given read count for total control (orange) and targeting (blue) sgRNA of an undifferentiated replicate. Normal distribution is overlaid (red). All data are representative of all samples sequenced for CRISPR screens.
Additional files
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Supplementary file 1
Gene expression profiles comparing day 0 and day 2 post estrogen withdrawal for Cas9+ER-Hoxb8 cells.
- https://cdn.elifesciences.org/articles/82289/elife-82289-supp1-v2.xlsx
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Supplementary file 2
MAGeCK RRA scores for genes positively and negatively selected during differentiation of Cas9+ER-Hoxb8 progenitors.
- https://cdn.elifesciences.org/articles/82289/elife-82289-supp2-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/82289/elife-82289-transrepform1-v2.pdf
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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
