Sea Pansy

Cas9 sequences were identified by Blast^{42 (link)} or obtained from published studies^{24 (link)–26} . Cas9 nuclease activity was inactivated by making the homologous D10A and H840A (S. pyogenes numbering) mutations in the RuvC and HNH nuclease domains for each Cas9 protein. dcas9 alleles were then codon optimized for mycobacterial expression with Jcat^{43 (link)} and synthesized by Genscript. dCas9 protein sequences used in this study are listed in Supplementary Table 1. Single-plasmid platforms were generated for all dcas9 alleles that contained: 1) the dcas9 allele under the control of a Tet repressor (TetR)-regulated UV15-Tet promoter^{1 (link)} or an optimized, synthetic TetR-regulated promoter (Supplementary Figure 4, Supplementary Table 1); 2) the cognate sgRNA under the control of a minimal synthetic constitutive promoter or an optimized, synthetic TetR-regulated promoter (Supplementary Figure 4, Supplementary Table 1); 3) a Tet repressor; 4) a single-copy L5-integrating backbone^{44 (link)}; 5) a pBR322-derived E. coli replication origin; and 6) a kanamycin-selectable marker. sgRNA scaffold sequences were obtained from published studies^{25 ,26} or engineered by fusing the crRNA direct repeat to the tracrRNA in an analogous manner to the S. pyogenes sgRNA^{11 (link)}. We attempted to enrich for highly active dCas9_Spy sgRNAs by selecting top-scoring sgRNAs based on the algorithm described in Doench et al.^{45 (link)}. All sgRNA scaffold sequences used in this study are listed in Supplementary Table 1.
To clone sgRNA targeting sequences, the sgRNA scaffolds were designed with two unique BsmBI restriction sites immediately 5’ to the sgRNA scaffold sequence. Complementary sgRNA targeting oligos (N20–25) were then annealed and ligated (T4 DNA ligase, NEB) into the BsmBI-digested CRISPRi vector backbone. To clone multiple sgRNAs into the same vector, the CRISPRi vector backbones were designed with a SapI-based Golden Gate cloning site 3’ to the first sgRNA scaffold. sgRNA targeting sequences are listed in Supplementary Table 2.
To construct the Renilla luciferase reporter plasmid, the Renilla gene was cloned downstream of the G13 promoter from Mycobacterium marinum^{46 (link)} with a synthetic 5’ untranslated region (UTR) in a single-copy Giles integrating vector^{47 (link)}. The Renilla 5’UTR was engineered such that the optimal PAM for each dCas9 protein could be tested with an identical sgRNA targeting sequence (UTR1) for all dCas9 proteins. The Renilla reporter sequences used for each dCas9 protein are shown in Supplementary Table 1.

Cell lines possessing the ecotropic retroviral receptor and the tetracycline repressor were generated and used in RNA interference library screening, shRNA toxicity assays and cDNA complementation studies as described4 (link). DLBCL cell lines were assigned to the ABC or GCB subtypes by gene expression profiling (ref.4 (link); Supplemental Fig. 14). shRNA screening results are given in Supplemental Tables 1 and 3, and shRNA sequences are listed in Supplemental Tables 2 and 3. Specific shRNA-mediated mRNA and protein knockdown was documented (Fig. 2C; Supplemental Fig. 6A, 15). IKK reporter lines were engineered to express an IκBα-Photinus luciferase fusion and Renilla luciferase27 (link). TIRF imaging of the BCR was based on previously described techniques12 .
Tumor biopsies were obtained prior to treatment from patients with de novo DLBCL28 (link), gastric MALT lymphoma, and Burkitt’s lymphoma. All samples were studied according to a protocol approved by the National Cancer Institute Institutional Review Board.