Journal: The Journal of Experimental Medicine
Article Title: In vivo CRISPR/Cas9 screens identify new regulators of B cell activation and plasma cell differentiation
doi: 10.1084/jem.20250594
Figure Lengend Snippet: 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 . (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 log 2 FC 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 ). 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 . The common positive regulators are shown in , as being significant in both cell types, while the PB-specific positive regulators are indicated as being nonsignificant in the GC B cell analysis . (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 ( [TD] and S1D [TI]), and the splenic mCherry + PBs, GC B cells, and memory B cells at day 7 ( [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.
Article Snippet: CD43 – B cells were enriched from the spleen or lymph nodes of mice by immunomagnetic depletion of non-B cells using CD43 (Ly-48) MicroBeads (Miltenyi Biotec).
Techniques: Sequencing, Infection, Cell Culture, Amplification, CRISPR, Cell Analysis, Virus, In Vivo, Clone Assay, Plasmid Preparation, Biomarker Discovery, Binding Assay, DNA Sequencing
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![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 . (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 log 2 FC 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 ). 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 . The common positive regulators are shown in , as being significant in both cell types, while the PB-specific positive regulators are indicated as being nonsignificant in the GC B cell analysis . (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 ( [TD] and S1D [TI]), and the splenic mCherry + PBs, GC B cells, and memory B cells at day 7 ( [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.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_0638/pmc12850638/pmc12850638__jem_20250594_figs2.jpg)