Description and characterization of different aspects of the sgRNA screening system. (A) Flow cytometric analysis of mCherry expression in B cells at 2 and 3 days after infection with a control mCherry-LV. The cells were cultured on OP9 cells with sBAFF (right). The frequencies of mCherry⁺ B cells are shown as mean values with SEM and were analyzed by one-way ANOVA with Tukey’s multiple comparisons test; ***P < 0.001; ****P < 0.0001. (B) Flow cytometric analysis of dividing and nondividing mCherry⁺ B cells after 3 days in culture on OP9 cells with sBAFF. Mature CD43^– B cells were incubated with CellTrace Violet prior to infection with a control mCherry-LV and were subsequently cultured for 3 days. The frequencies of mCherry⁺ B cells among the dividing and nondividing B cell population are shown as mean values with SEM and were analyzed by the unpaired t test; ***P < 0.001. (C) Immunofluorescence staining of spleen sections from recipient mice at 5 and 7 days after immunization with NP-KLH/alum. LV infection and B cell transfer were performed as described in D. Spleen sections were stained with antibodies against IgD (green), mCherry (red), TCRβ (blue), and IRF4 (white). A nonimmunized mouse, which did not receive transduced mCherry⁺ B cells, was used as a control. The scale bar represents 100 μm. (D) Time course of plasmablast formation upon immunization with NP-Ficoll. Naïve B cells isolated from the donor mice were transduced with LV particles expressing a neutral control sgRNA (sg.Chr1) and the mCherry reporter protein. After infection, B cells were cultured in vitro for 3 days in the presence of stromal OP9 cells and sBAFF. Transduced mCherry⁺ B cells were isolated and transferred to recipient mice, which were immunized 16 h later with NP-Ficoll in PBS. Flow cytometric analysis of splenocytes from recipient mice at day 3, 5, and 7 after immunization (left) revealed the percentages of mCherry⁺ B cells in total splenocytes and mCherry⁺ plasmablasts (TACI⁺CD138⁺) within the mCherry⁺ cells, as shown in the bar graphs (right). Absolute numbers of the mCherry⁺ B cells and mCherry⁺ plasmablasts in the spleen are also indicated (far right). Statistical data are shown as mean values with SEM and were analyzed by the Welch ANOVA with the Brown–Forsythe test; *P < 0.05; **P < 0.01; ****P < 0.0001. Each dot represents one mouse. AF, autofluorescence measured in the BV605 channel. (E) Scheme describing the steps taken for the selection of the 379 genes to be studied in the in vivo CRISPR/Cas9 screening experiments. The data shown in A, B, and D are based on two independent experiments. Each dot corresponds to one mouse.