CRISPR/Cas9 screen for identifying new regulators of the TI B cell responses. (A) Schematic diagram of the in vivo CRISPR/Cas9 screening experiments used to study the TI B cell responses. The TI screening experiment was performed as described in detail for the TD screening experiment (Fig. S3 A), except that the transplanted mice were immunized with NP-Ficoll and that plasmablasts (TACI⁺CD138⁺) and memory B cells (Mem B, TACI⁺CD138⁻) were isolated at day 6 or 7 by flow cytometry. (B) Flow cytometric analysis of enriched mCherry⁺ CD45.2⁺ B cells from recipient mice. The gates used for the sorting of plasmablasts and memory B cells are indicated. The percentages of mCherry⁺ plasmablasts and mCherry⁺ memory B cells within the sorted cell population are shown. (C) Numbers of sorted plasmablasts and memory B cells (left), the percentage of mCherry⁺ plasmablasts and mCherry⁺ memory B cells within the sorted cell population (middle), and the numbers of sorted mCherry⁺ plasmablasts and mCherry⁺ memory B cells (right) were determined for individual recipient mice analyzed in two independent screening experiments. (D) Total sgRNA representation in each plasmablast and memory B cell replicate sample analyzed in two screening experiments. The sgRNA representation was estimated by dividing the number of mCherry⁺ plasmablasts or mCherry⁺ memory B cells by the number of the 882 sgRNAs constituting the sgRNA library. Each plasmablast and memory B cell replicate sample contained cells that were obtained from seven or nine recipient mice after pooling. Each colored box represents the individual contribution of each mouse to the sgRNA representation of the entire replicate sample. (E) Flow cytometric definition of mCherry⁺ memory B cells. Sorted mCherry⁺ donor B cells were transferred to recipient mice, which were immunized 16 h later with NP-Ficoll in PBS. Flow cytometric analysis of splenocytes 6 days after immunization is shown. Donor-derived mCherry⁺ cells were mainly TACI⁺CD138⁺ plasmablasts or CD138⁻GL7⁻CD38⁺TACI⁺ cells that phenotypically correspond to memory B cells.