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Supporting Information Supporting Information Chu et al. 10.1073/pnas.1613884113 Antibodies (Ter-119), anti-Ckit (2B8), anti-Sca1 (D7), and anti-CD64 (clone The following anti-mouse antibodies were purchased from X54-5/7.1). Anti-CD95 (15A7), anti-CD14 (clone Sa2-8), and anti- Biolegend: anti-B220 (RA3-6B2), anti-CD19 (6D5), anti-CD138 Tnfrsf13b (ebio8F10-3) antibodies were ordered from eBioscience. (281-2), anti-CD22 (OX-97), anti-CD24 (M1/69), anti-CD44 Anti-Cas9 (7A9-3A3) antibody was purchased from Cell Signaling (IM7), anti-CD80 (16-10A1), anti-CD81 (Eat-2), anti-CD83 Technology, and anti-Flag (M2) antibody and anti–β-actin were (Michel-19), anti-CD86 (GL-1), anti-CD45 (30-F11), anti-CD11b purchased from Sigma. HRP-conjugated secondary anti-mouse Ig (M1/70), anti-Gr-1 (RB6-8C5), anti-CD3e (145-2C11), anti-Ter-119 antibody was ordered from Rockland Immunochemicals. A In the mLN B cells Myeloid cells T cells C57BL/6 R26-Cas9iGFP/+ CD3 CD19 CD11b Cas9-iGFP B CD19+B220+ splenic B cells B2 B cells B1 B cells C57BL/6 B1: 4.1 R26-Cas9iGFP/+ CD43 CD19 B2: 95 CD5 Cas9-iGFP CD19+ peritoneal B cells B220loCD43+ B1 B2 B cells B1a B cells B1b B cells B1: 80 B1a: 64 CD5 CD19 CD43 B2: B1b: 20 31 B220 IgM Cas9-iGFP C Spleen CD19+B220+ CD38loFashi GC 55.4 CD19 CD38 CD19 1.2 B220 Fas Cas9-iGFP mLN 40.1 C57BL/6 R26-Cas9iGFP/+ CD19 CD38 3.8 CD19 B220 Fas Cas9-iGFP + + + Fig. S1. Characterization of heterozygous R26-Cas9iGFP transgenic mice. (A) FACS plots of T cells (CD3 ), myeloid cells (CD11b ), and B cells (CD19 ) isolated from the mLNs of Cas9 transgenic or wild-type animals. (B) FACS plots of B cells (CD19+) isolated from the peritoneal cavity and spleen of Cas9 transgenic or wild-type animals. Gates were set on B1 and B2 cells in the spleen and B2, B1a, and B1b in the peritoneal cavity based on CD19, B220, CD43, CD5, and IgM + + markers as indicated. (C) FACS plots of B220 CD19 FashiCD38low GC B cells isolated from the spleen and mLNs. Data are representative of four independent experiments. Cells from wild-type C57BL/6 animals (red) and R26-Cas9iGFP/+ animals (blue) are shown in the same FACS plots. Chu et al. www.pnas.org/cgi/content/short/1613884113 1of9 A BM cells Ckit+Sca1+ LSK 2.7 C57BL/6 Lin Ckit R26-Cas9p2aGFP/+ 17 Max of % R26-Cas9iGFP/+ CD45 Sca1 Cas9-GFP Monocytes B CD19-CD3- B cells T cells Granulocytes Dendritic cells B cells: 21.4 Mono/Dendritic 19.1 T cells BM 2.5 Granulocytes 65 B cells: 63.4 Mono/Dendritic 46.6 T cells Spleen 26.5 Granulocytes CD19 CD11b 27.6 CD3 Gr-1 Max of % Cas9-GFP − + + Fig. S2. Comparison of GFP expression levels in HSCs of R26-Cas9iGFP and R26-Cas9p2aGFP mice. (A) FACS plots of HSCs (defined as Lin Sca1 c-Kit or LSK), + + isolated from the BM of R26-Cas9iGFP/+ (blue), R26-Cas9p2aGFP/+ (red), and C57BL/6 (black) mice. (B) FACS plots of B cells (CD19 ), T cells (CD3 ), granulocytes (Gr1+CD11b+), and monocytes/dendritic cells (CD11b+Gr1−) isolated from the BM and spleen of R26-Cas9iGFP (blue), R26-Cas9p2aGFP (red), and wild-type (black) mice. The data are representative of two independent experiments. Chu et al. www.pnas.org/cgi/content/short/1613884113 2of9 A α-CD40/IL-4 stimulated B cells BMDMs C57BL/6 R26-Cas9iGFP/+ C57BL/6 R26-Cas9iGFP/+ Flag (M2) Flag (M2) Cas9 Cas9 Actin Actin μ WCL: 30μg/lane WCL: 10 g/lane B C57BL/6 R26-Cas9iGFP/+ Flag tag (Cas9) DAPI Flag tag (Cas9) DAPI C sgCD38-1 sgCD38-2 sgFas-1 sgFas-2 D α-CD40/IL-4 stimulated B cells R26-Cas9iGFP/+ R26-Cas9p2aGFP/+ #356 #357 #1 #2 C57BL/6 58 57 38 91 Cas9 Actin 19 22 8 54 Fas CD38 GFP R26-Cas9p2aGFP/+ R26-Cas9iGFP/+ Fig. S3. Cas9 levels and localization in primary cells of R26-Cas9iGFP/+ mice and Cas9-dose-dependent knockout efficiency. (A) Western blots of Cas9 proteins (detected by anti-Cas9 and anti-Flag antibodies) in anti-CD40/IL-4–activated B cells and BMDMs isolated from R26-Cas9iGFP/+ and wild-type animals. β-Actin was used as a loading control. (B) Immunostaining of Cas9 in BMDMs derived from R26-Cas9iGFP/+ and wild-type animals. Cas9 was stained using anti-Flag (M2) antibodies (red). DAPI was used for counterstaining of nuclei (green). (C) Knockout efficiencies in B cells from R26-Cas9iGFP/+ (Top) and R26-Cas9p2aGFP/+ (Bottom) mice that were activated and transduced with retroviruses expressing sgCD38-1, sgCD38-2, sgFas-1, and sgFas-2 side by side. The gates in the FACS plots show the percentage of cells that lost the surface marker 4 d after transduction. (D) Western blots of Cas9 proteins in anti-CD40/IL-4–activated B cells isolated from R26-Cas9iGFP/+, R26-Cas9p2aGFP/+, and wild-type animals. The data are representative of two independent experiments. Chu et al. www.pnas.org/cgi/content/short/1613884113 3of9 Fig. S4. Knockout efficiencies in Cas9-expressing B cells. (A) Selection of sgRNAs designed with CrispRGold targeting 12 B-cell surface makers. Targeting sequences are shown 5′–3′.(B) FACS plots showing the knockout efficiencies in Cas9-expressing B cells 4 d after transduction with sgRNAs against the indicated surface markers. Note that some surface markers are only induced upon activation (CD83 and Tnfrsf13b). The sgRNAs used are indicated in the title of each FACS plot. The data are representative of three independent experiments. Chu et al. www.pnas.org/cgi/content/short/1613884113 4of9 A R26-Cas9iGFP/+ 70% Retroviral transduction T cells FACS α-CD3+ α-CD3+ Puro C57BL/6 α-CD28 α-CD28 30% (4 days) (2 days) (1 day) B Lentiviral transduction R26-Cas9iGFP/+ BM cells FACS M-CSF Puro (14 days) (4 days) C57BL/6 BM cells FACS M-CSF Puro (14 days) (4 days) Bone marrow derived macrophages (BMDMs) C sgCD64-1 sgCD14-1 2.37 3.91 C57BL/6 100 80 60 40 54.2 44.6 R26-Cas9iGFP/+ Knockout (%) Knockout 20 sgRNA-1 sgRNA-2 0 CD64 CD14 FSC CD64 CD14 Fig. S5. CRISPR-mediated gene knockout in primary T cells and BMDMs. (A) Cas9-expressing T cells were mixed with wild-type T cells. The cells were activated with anti-CD3 and anti-CD28 antibodies for 2 d. The activated T cells were transduced with retroviral particles expressing sgRNAs. The transduced T cells were selected with puromycin for 4 d before assessment of knockout efficiencies. (B) Scheme of CRISPR-mediated gene knockout in primary BMDMs. BM cells from R26-Cas9iGFP/+ mice and wild-type mice were differentiated for 2 wk in the presence of M-CSF. BMDMs were transduced with lentiviruses expressing sgRNAs targeting mouse CD64 and CD14 genes. The transduced cells were selected with puromycin for 4 d and the gene knockout efficiency was analyzed by flow cytometry. (C) FACS plots of the transduced BMDMs derived from control animals (Top) and R26-Cas9iGFP/+ animals (Bottom) stained with antibodies against the targeted surface markers. The summary of the experiments is shown at Right. The data are representative of three individual Cas9 transgenic mice (each representing a data point in the summary). Chu et al. www.pnas.org/cgi/content/short/1613884113 5of9 Fig. S6. Robust detection of transcription factors important for B-cell activation and differentiation. (A) Scheme of B-cell activation and differentiation. + Splenic B cells were activated with anti-CD40 IL4 and subsequently differentiated into plasmablasts using IL-21 and CD40-ligand expressing feeder cells. + − (B) sgRNAs used to target Prdm1, Xbp1, Pou2af1, Irf4, and Myc.(C) Analysis of the percentage of GFP Cas9-expressing B cells and GFP wild-type B cells. These cells were mixed, activated, and transduced (individually) with three sgRNAs per gene targeting Prdm1, Xbp1, Pou2af1, Irf4,andMyc. The FACS plots show the GFP+ Cas9-expressing B cells 4 and 8 d after transduction. (D) Analysis of the induction of CD138 in GFP+ Cas9-expressing and GFP− wild-type B cells 6 d after transduction. The data are representative of three independent experiments. Chu et al. www.pnas.org/cgi/content/short/1613884113 6of9 Color Key and Histogram Count 01025 −2 −1 0 1 2 Row Z−Score GC B cells Plasmacell_Spleen Plasmacell_BM FO.B cells Atf3 Atf4 Atf6 Klf2 Klf4 Klf6 Brd2 Brd4 Calm1 Calm2 Ccnd2 Ost4 Fam46c Cdkn2c Cebpb Ostc Pim1 Pim2 Pik3cd Pik3r1 Egr1 Egr2 Fli1 Fos Fosb Irak1 Irak2 Irf1 Irf2 Irf3 Irf4 Jun Junb Jund1 Mcl1 Mef2a Mef2c Myc Pdcd5 Pou2af1 Pou6f1 Prdm1 Prdx1 Prdx2 Prdx3 Prdx4 Preb Prkcd Prkd2 Rab1 Rabac1 Stat3 Stat5b Tra1 Tmem176a Tmem176b Xbp1 S100a8 S100a9 Creld2 Zfp36 Tnfrsf17 Tnfrsf13b Rhob Tcf4 Sox4 Myb Tcl1b3 Mzb1 Arf4 Pomp Edem1 Dis3 Slc44a1 Rftn1 Neat1 Atat1 Os9 Mia3 Emc7 Ndufv3 Qrich1 Hid1 Fig. S7. Gene set used for the small-scale screen. Total RNA was isolated from follicular B, GC, and plasma cells that were isolated from the spleen and BM of immunized animals. Microarrays were performed and data were normalized before analysis. The heatmap shows the expression levels of the selected genes with differential expression in the plasma cell populations. Chu et al. www.pnas.org/cgi/content/short/1613884113 7of9 A Gene 1 Gene 2 Gene 3 Gene 4 Gene 5 Gene 6 Gene 7 Gene 8 Gene 9 ... sgRNAs Plasmid sgRNA mini-library plate Cloning Retroviral sgRNA Retrovirus production mini-library plate Cas9 and WT B cell plate Target cell transduction Transduced cell Selection plate Cell phenotype-HTS-FACS Repressor genes Repressor genes Survival/ Plasma cell proliferation differentiation Expansion index Diffentiation index Diffentiation Enhancer genes Enhancer genes Expansion index Differentiation index B Survival and proliferation Dis3, Egr2, Preb, Fosb, Pik3r1 12 Ccnd2, Pomp, Pdcd5, S100a8 Stat3, Klf2, Cdkn2c Input: sgRNA screening 83 gene candidates Xbp1, Pou2af1, Myc, Irf4, 13 gene controls 10 Ost4, Junb, Hsp90b1, Prkd2, Klf6, Edem1 8 Prdm1, Arf4, Pim2, Brd4, Creld2, Calm1, Irf1, Zfp36 Plasma cell differentiation Fig.
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