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:

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

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 muie nml.Mcoraswr efre n aawr omlzdbfr nlss h eta hw h xrsinlvl fteslce gen selected the of levels expression the shows heatmap populations. The cell analysis. plasma before the normalized in were expression data and differential performed with were Microarrays animals. immunized S7. Fig. h tal. et Chu eestue o h ml-cl cen oa N a sltdfo olclrB C n lsaclsta eeioae rmtesle n Mof BM and spleen the from isolated were that cells plasma and GC, B, follicular from isolated was RNA Total screen. small-scale the for used set Gene www.pnas.org/cgi/content/short/1613884113

Count 01025

FO.B 2 cells 1 0 −1 −2 and Histogram Row Z−Score Color Key CBclsPamcl_penPlasmacell_BM Plasmacell_Spleen GC Bcells Egr1 Pik3r1 Pik3cd Pim2 Pim1 Ostc Cebpb Cdkn2c Fam46c Ost4 Ccnd2 Calm2 Calm1 Brd4 Brd2 Klf6 Klf4 Klf2 Atf6 Atf4 Atf3 Hid1 Qrich1 Ndufv3 Emc7 Mia3 Os9 Atat1 Neat1 Rftn1 Slc44a1 Dis3 Edem1 Pomp Arf4 Mzb1 Tcl1b3 Myb Sox4 Tcf4 Rhob Tnfrsf13b Tnfrsf17 Zfp36 Creld2 S100a9 S100a8 Xbp1 Tmem176b Tmem176a Tra1 Stat5b Stat3 Rabac1 Rab1 Prkd2 Prkcd Preb Prdx4 Prdx3 Prdx2 Prdx1 Prdm1 Pou6f1 Pou2af1 Pdcd5 Myc Mef2c Mef2a Mcl1 Jund1 Junb Jun Irf4 Irf3 Irf2 Irf1 Irak2 Irak1 Fosb Fos Fli1 Egr2 7of9 es 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 Expansion 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. S8. Small-scale CRISPR-mediated screening to detect novel genes important for B-cell activation and plasma cell differentiation. (A) Scheme of the high- throughput 96-well-based screening approach. One sgRNA was designed for each gene of interest. Each sgRNA was separately cloned into a retroviral vector. The cloning, retrovirus production, and transduction of B cells were performed in 96-well plates. The transduced cells were selected with puromycin. Survival/ proliferation was analyzed based on monitoring the percentage of GFP+ Cas9-expressing B cells at different time points. The fold changes were calculated by comparing to the percentage of GFP+ cells at day 2, allowing for the identification of repressor or enhancer genes. The plasmablast assay was based on the + + − + normalization of GFP -derived CD138 plasmablasts to GFP -derived CD138 plasmablasts. (B) Diagram showing the screen set and identification of important genes for survival and proliferation (Middle, Top) and plasma cell differentiation (Middle, Bottom). A total of 22 genes were crucial for activated B-cell survival and proliferation, and 8 genes were essential for plasma cell generation.Ten genes were essential for both survival/proliferation and differentiation.

Chu et al. www.pnas.org/cgi/content/short/1613884113 8of9 A sgRosa26 sgMyc-3 sgEgr2 sgDis3 sgOst4 sgPreb sgPomp

59.2 40.8 60.1 39.9 64.1 35.9 62.3 37.7 55.1 44.9 62.3 37.7 60.9 39.1 Day 2

59.9 40.3 86.0 14.0 89.3 10.8 83.8 16.4 68.1 32.5 72.8 27.8 74.0 26.0 Day 4

60.0 40.0 90.1 9.9 95.9 4.1 85.7 14.4 82.9 17.1 85.6 14.4 83.6 16.4 Day 6

60.8 39.2 93.1 6.9 97.6 2.4 93.3 6.7 87.3 12.7 93.2 6.8 89.3 10.7

Day 8 FSC Cas9-iGFP B sgRosa26 sgPrdm1-3 sgOst4 sgArf4 sgCreld2 sgZfp36 sgEdem1 0.79 0.37 0.44 0.26 1.20 0.43 0.06 0.03 1.10 0.85 0.42 0.21 1.22 1.04 Day 2

60.4 38.5 60.6 38.7 61.8 36.6 62.7 37.2 59.5 38.8 60.7 38.7 58.3 39.4 16.2 13.6 17.1 0.96 21.0 0.88 23.0 3.11 19.8 36.0 14.0 26.0 14.0 26.6 Day 6

CD138 39.0 31.2 48.6 33.3 60.6 17.5 52.5 21.4 25.5 18.7 34.6 25.5 22.7 36.6 Cas9-iGFP

+ Fig. S9. Validation of candidates important for B-cell survival/proliferation and plasma cell differentiation. (A) FACS data showing the percentage of GFP − Cas9-expressing and GFP wild-type B cells 2, 4, 6, and 8 d after transduction with the indicated sgRNAs. As before, Cas9-expressing B cells were mixed with wild-type B cells, activated, and transduced with retroviral particles expressing sgRNA targeting Rosa26 (as control), Myc, Egr2, Dis3, Ost4, Preb, and Pomp. (B) Analysis of the induction of CD138 in GFP+ Cas9-expressing and GFP− wild-type B cells. Cas9-expressing B cells were mixed with wild-type B cells, activated, and transduced with retroviral particles expressing sgRNAs targeting Rosa26 (as control), Prdm1, Ost4, Arf4, Creld2, Zfp36, and Edem1.

Chu et al. www.pnas.org/cgi/content/short/1613884113 9of9