Validation of positive and negative regulators of plasmablast and GC B cell formation by sgRNA-mediated gene inactivation. (A) mCherry⁺ and mAmetrine⁺ sgRNA-expressing B cells were mixed at a 2.5:1 ratio and transferred into recipient mice, followed by NP-KLH immunization. Flow cytometric analysis of splenocytes from recipient mice was performed 7 days after immunization. mCherry⁺ B cells expressed either the control sgRNA (sg.Chr1) or sgRNAs targeting Prdm1 (sg.Prdm1), positive regulator genes (sg.Manf and sgZyve21), or negative regulator genes (sg.Prdx4 and sg.Atxn1). The percentage of mCherry⁺ (red gate) and mAmetrine⁺ (blue gate) B cells among the total B cells (CD19⁺CD138^–/lo and CD19^loCD138⁺; green gate) is shown. The percentages of mCherry⁺ and mAmetrine⁺ plasmablasts (TACI⁺CD138⁺) and GC B cells (GL7⁺CD95⁺) in total B cells (green numbers) and within the mCherry⁺ B cells (red numbers) or in mAmetrine⁺ B cells (blue numbers) are indicated. (B and C) Analysis of the loss (left) or gain (right) of plasmablasts and GC B cells upon sgRNA-mediated inactivation of candidate genes coding for positive or negative regulators, respectively. The ratio of the percentage of mCherry⁺ plasmablasts (B) or mCherry⁺ GC B cells (C) (expressing the sg.Control [sg.Chr1] or sgRNAs targeting the indicated genes) versus mAmetrine⁺ plasmablasts (B) or mAmetrine⁺ GC B cells (C) (expressing the sg.Control) was determined in total B cells, respectively. The calculation of the different ratios is explained in detail in Fig. S5 C. The ratios, which were determined by analyzing different control mice (gray), experimental mice (black), and mice with inactivation of the known regulator genes Prdm1 or Irf4 (red), are indicated. Statistical data are shown as mean values with SEM and were analyzed by the multiple unpaired t test with Holm–Šídák’s multiple comparisons test; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Each dot represents one mouse. The validation experiments were performed at least two times.