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. Author manuscript; available in PMC: 2019 Oct 3.
Published in final edited form as: Nature. 2019 Apr 3;568(7751):187–192. doi: 10.1038/s41586-019-1088-4

Figure 1. Combined CRISPR-Cas9 screens and RNA-seq analysis identify an age-associated genetic modifier of phagocytosis.

Figure 1.

a, Screening strategy for age-related genetic modifiers of phagocytosis. Scale bar = 50 microns. Screens were performed in technical duplicate.

b, RNA-seq differential expression analysis between aged (20 m.o., n=6) and young (3 m.o., n=6) primary microglia overlaid with significant hits from CRISPR-Cas9 screen of BV2 cells. Red dots represent hits that promote phagocytosis, blue dots represent hits that inhibit phagocytosis, and gray dots are knockouts that showed no effect. Dashed line represents adjusted P-value of 0.001 (Benjamini-Hochberg method).

c, qPCR analysis of CD22 expression in acutely isolated primary microglia from young (2-3 m.o.) and aged (20-22 m.o.) mice, normalized to a housekeeping gene (β-actin); data represent fold change relative to young microglia (n=4, ****P<0.00005, two-sided t-test, mean +/− s.e.m.).

d, Flow cytometry quantification (mean fluorescence intensity (MFI) – fluorescence minus one (FMO) background intensity) of CD22 expression in acutely isolated primary microglia from young (2-3 m.o.) and aged (20-22 m.o.) mice (n=5, ***P<0.0005, two-sided t-test, mean +/− s.e.m.).

e, Normalized phagocytosis (fluorescent area / confluence) monitored over 24 hours, imaged every hour. Control BV2 cells were infected with a “safe-targeting” sgRNA and KO BV2 cells were infected with a sgRNA targeting CD22 (n=6, ****P<0.00005, two-sided t-test; mean +/− s.e.m.).

f, Representative images of young and aged cerebella probed for Tmem119 (green), CD22 (red), and nuclei (DAPI, blue). Green arrows indicate Tmem119+ microglia, and red-green arrows indicate Tmem119+CD22+ microglia. Scale bar = 30 microns.

g, Percentage of Tmem119+ microglia that co-express CD22 in multiple brain regions of young (3 m.o., brown) and aged (22 m.o., gray) mice (n=3, *P<0.05, ***P<0.0005, ****P<0.00005, 2-way ANOVA with Sidak correction, mean +/− s.e.m.).

Data in c-g were replicated in at least 2 independent experiments.