Testing of the in vivo model system used for CRISPR/Cas9 screening experiments. (A) Schematic diagram of the setup used for testing the in vivo CRISPR/Cas9 screening system. Naïve B cells isolated from donor mice were transduced with LV particles expressing a neutral control sgRNA and the mCherry reporter protein. After infection, B cells were cultured in vitro for 3 days in the presence of OP9 cells and sBAFF. Transduced mCherry⁺ B cells were sorted by flow cytometry and transferred to recipient mice, which were immunized 16 h later with NP-KLH in alum. mCherry⁺ plasma cells and mCherry⁺ GC B cells were analyzed by flow cytometry at different time points after immunization. (B) Naïve B cells of the donor genotype were infected with a mCherry-LV expressing a neutral control sgRNA and sorted followed by transfer to mice of the recipient genotype and NP-KLH immunization, as described in A. Flow cytometric analysis of splenocytes from recipient mice was performed 3, 5, 7, and 10 days after immunization. The percentages of total mCherry⁺ B cells in total splenocytes, mCherry⁺ plasmablasts (TACI⁺CD138⁺), and mCherry⁺ GC B cells (GL7⁺CD95⁺) within the mCherry⁺ B cell population (left), and the absolute numbers of these cells (right) are indicated. AF, autofluorescence measured in the BV605 channel. (C) Naïve B cells isolated from donor mice were transduced with LV particles expressing a control sgRNA (sg.Chr1) or sgRNAs targeting Prdm1 or Irf4. After infection, B cells were cultured in vitro for 3 days and transduced mCherry⁺ B cells were transferred to recipient mice followed by immunization with NP-KLH, as described in A. 6 days after immunization, the splenocytes were analyzed by flow cytometry. The percentages of total mCherry⁺ B cells, mCherry⁺ plasmablasts, and mCherry⁺ GC B cells among total B cells are indicated. Statistical data (B and C) are shown as mean values with SEM and were analyzed by the multiple unpaired t test with Holm–Šídák’s correction test; *P < 0.05; **P < 0.01; ****P < 0.0001. Two independent experiments (B and C) were performed. Each dot represents one mouse.