Indel sequencing data, identification of GC B cell– and PB-specific sgRNA hits, and sgRNA representation at different stages of the screening experiments. (A and B) Indel sequencing. Mature CD43^– B cells were infected with the indicated sgRNA mCherry-LVs, cultured for 3 days on OP9 cells with sBAFF, and then stimulated with CpG, IL-4, and IL-5 for another 3 days, followed by flow cytometric sorting of the mCherry⁺ B cells and DNA preparation. Indel sequencing was performed by PCR amplification and sequencing of a DNA fragment spanning the sgRNA break site, followed by TIDE analysis (Brinkman et al., 2014). (A) Percentages of indels are indicated relative to the break site of sg.Cd44 (position 0, black), demonstrating that the percentage of indels is 88% for the sg.Cd44. (B) Percentages of indels are shown for the indicated sgRNAs, as determined by the indel sequencing and TIDE analysis. (C) Identification of GC B cell–specific and PB-specific positive and negative regulators, as determined by CRISPR/Cas9 screening experiments at day 7 after NP-KLH immunization. The log₂FC plot (left) indicates the sgRNA hits that were determined by a more than twofold change in sgRNA abundance in GC B cells and PBs (corresponding to Fig. 4, B–E). The genes corresponding to the GC B cell–specific and PB-specific sgRNA hits are shown (right), and their fold changes and P values are indicated in Table S3. The common positive regulators are shown in Fig. 4, C and D, as being significant in both cell types, while the PB-specific positive regulators are indicated as being nonsignificant in the GC B cell analysis (Fig. 4, C and D). (D) sgRNA representation at different stages of the TD and TI screening experiments. Flow cytometric analysis was used to determine the number of sorted mCherry⁺ B cells at the start, the splenic mCherry⁺ B cells at day 3 after B cell transfer (Fig. 3 B [TD] and S1D [TI]), and the splenic mCherry⁺ PBs, GC B cells, and memory B cells at day 7 (Figs. S3 D [TD] and S4D [TI]). As the screening library contained 882 sgRNAs, the number of identified B cells was divided by 882 to determine how many cells contained one specific sgRNA (cells/sgRNA) under the assumption that each cell was only infected by one sgRNA virus (MOI = 1). (E) Schematic representation of the LV vectors used in this study. LVs containing the PGK-mCherry or EF1a-mCherry gene were used for establishing and testing of the in vivo CRISPR/Cas9 screening system. The sgRNA library was cloned in a LV vector containing the EF1as-mCherry gene. The validation experiments were performed with LVs containing the EF1as-mCherry or EF1as-mAmetrine gene. 5′LTR, 5′ long terminal repeat; Ψ, psi packaging signal; RRE, Rev response element; cPPT, central polypurine tract; PBS, primer binding site for DNA sequencing library preparation; hU6, human U6 promoter; hPGK, human phosphoglycerate kinase promoter; hEF1a, human elongation factor 1a promoter; hEF1as, human elongation factor 1a short promoter; WPRE, woodchuck hepatitis virus posttranscriptional regulatory element; 3′LTR (SIN), 3′ long terminal repeat (self-inactivating); FC, fold change; PBs, plasmablasts.