CRISPR/Cas9 screens for new regulators of B cell responses. (A–E) Results of the CRISPR/Cas9 screens performed upon TD immunization. (A) Difference of sgRNA abundance in PBs versus donor B cells (left) and GC B cells versus donor B cells (right) is shown as log₂ FC for all sgRNAs in the library, neutral control sgRNAs targeting olfactory receptor genes (Olfr), sgRNAs targeting essential genes, and sgRNAs targeting the well-known PB regulators Irf4 and Prdm1. (B) Volcano plot displaying the depletion or enrichment of sgRNAs (x axis) in PBs versus donor B cells (left) and in GC B cells versus donor B cells (right). The P values (y axis) were calculated using MAGeCK (Li et al., 2014). For each gene, the sgRNA with the more significant P value was plotted. A twofold change in sgRNA abundance is indicated by a dashed line. Potentially positive (brown, P < 0.05, log₂FC ≤ −1) and negative (blue, P < 0.05, log₂FC ≥ 1) regulators are highlighted for PBs (left plot) and GC B cells (right plot). (C–E) Heat map showing the depletion or enrichment of sgRNAs in PBs versus control donor B cells (left column) and in GC B cells versus control donor B cells (right column). The different color shadings indicate the log₂FC, while the circle size refers to the P value (−log₁₀). Genes uniquely found in our in vivo screens for PB regulators are highlighted in green color. Erlec1, Qpctl, Zfyve21, and Isg20 were previously identified in an in vitro CRISPR/Cas9 screen as regulators of antibody secretion, but not as regulators of PB differentiation (Trezise et al., 2023). The sgRNA depletion data for well-known positive regulators of plasma cell development (above) and positive regulators involved in the homeostasis of the ER (below) are shown in C. The sgRNA depletion data of novel potentially positive regulators of PB differentiation or homeostasis are shown in D. The sgRNA enrichment data for novel potentially negative regulators of PB differentiation or homeostasis are shown in E. ns, nonsignificant. (F–H) Results of the CRISPR/Cas9 screens performed upon TI immunization. (F) Volcano plot displaying the depletion or enrichment of sgRNAs (x axis) in PBs versus donor B cells (left) and memory B cells (Mem B, TACI⁺CD138⁻) versus donor B cells (right). The P values (y axis) were calculated using MAGeCK. For each gene, the sgRNA with the more significant P value was plotted. Potentially positive (brown, P < 0.05 and log₂FC ≤ −1) and negative (blue, P < 0.05 and log₂FC ≥ 1) regulators are highlighted for PBs (left) and memory B cells (right). (G and H) Common potential PB regulators identified in the TI and TD CRISPR/Cas9 screening experiments. The heat maps show the depletion (G) or enrichment (H) of the sgRNAs, which were identified in the TI CRISPR/Cas9 screen by comparing PBs versus control donor B cells (left column) and memory B cells versus control donor B cells (right column). The sgRNA depletion data for well-known positive regulators of plasma cell development (left) and novel potentially positive regulators of PB differentiation or homeostasis are shown in G, while the respective data for potentially negative regulators are indicated in H. The Tnfrsf17 (BCMA) sgRNA was depleted only in the TI sgRNA screen. ns, nonsignificant. Multiple replicate samples were analyzed in two independent sgRNA screening experiments in response to TD or TI immunization, as described in detail in Fig. S3 D (TD) and Fig. S4 D (TI). FC, fold change; PB, plasmablast.