Validation of gene hits of the TD B cell responses by sgRNA-mediated gene inactivation. (A) Schematic diagram of the experiments used for validating potential regulators identified by the in vivo CRISPR/Cas9 screens. Naïve splenic B cells of the donor genotype were transduced with LV particles expressing the mCherry reporter protein and a sgRNA targeting the gene of interest. In parallel, donor B cells were transduced with LV particles expressing the mAmetrine reporter protein and the control sgRNA (sg.Chr1). After infection, the cells were cultured in vitro for 3 days in the presence of OP9 cells and sBAFF. Transduced mAmetrine⁺ and mCherry⁺ B cells were sorted by flow cytometry, mixed, and transferred to recipient mice, which were then immunized with NP-KLH/alum 16 h later. The frequency of mAmetrine⁺ and mCherry⁺ PBs in the spleen was analyzed by flow cytometry 7 days after immunization. (B) mCherry⁺ and mAmetrine⁺ sgRNA-expressing B cells were mixed at a 2.5:1 ratio and transferred into recipient mice, followed by 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.Amigo2, sg.Slc22a17, and sg.Qpctl), and a negative regulator gene (sg.Cd44). 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⁺ PBs (TACI⁺CD138⁺) and GC B cells (GL7⁺CD95⁺) in total B cells (green numbers) and within the mCherry⁺ B cells (red numbers) or mAmetrine⁺ B cells (blue numbers) are indicated. PB, plasmablast.