Journal: Nature immunology

Article Title: Multiplexed activation of endogenous genes by CRISPRa elicits potent anti-tumor immunity

doi: 10.1038/s41590-019-0500-4

Figure Lengend Snippet: a, Schematic of the experimental design for using CRISPRa to enhance the immune recognition of tumor-associated antigens (TAAs), eliciting systemic immune responses. b, c, E0771-dCas9-VP64 cells were transduced with lentivirus to express ovalbumin (OVA) under a PGK promoter (E0771-OVA), and further transduced with either Vector or CRISPRa sgRNAs targeting the PGK promoter. ( b), Representative flow cytometry analysis of surface staining for OVA-derived SIINFEKL-H-2K b complex on cells transduced with Vector or sgRNAs. (c), Mean fluorescence intensity (MFI) of APC-SIINFEKL-H-2K b on E0771-OVA cells transduced with Vector or sgRNAs. n = 15 cell replicates (SIINFEKL-H-2K b staining in Vector), n = 13 (isotype in Vector), n = 19 (SIINFEKL-H-2K b staining in CRISPRa sgRNAs), or n = 15 (isotype in CRISPRa sgRNAs) from four independent experiments. Two-sided Mann-Whitney test: SIINFEKL-H-2K b staining vs. isotype in Vector, p = 0.0356; SIINFEKL-H-2K b staining vs. isotype in CRISPRa sgRNAs, p < 0.0001; SIINFEKL-H-2K b staining in CRISPRa sgRNAs vs. Vector, p < 0.0001. d, The percentage of IFN-γ-producing OT-I CD8 + T effector cells after co-culture with the indicated E0771-OVA cancer cells for 3h. n = 11 co-culture samples from three independent experiments. Welch’s two-tailed unpaired t-test: CRISPRa sgRNAs vs. vector in E:T = 1:1, p = 0.0218; CRISPRa sgRNAs vs. vector in E:T = 0.5:1, p = 0.002. e, The percentage of viable cancer cells (excluding dead cells and apoptotic cells) when co-cultured with OT-I CD8 + T effector cells for 24h. n = 11 co-culture samples from 3 independent experiments. Welch’s two-tailed unpaired t-test: CRISPRa sgRNAs vs. vector in E:T = 1:1, p < 0.0001; CRISPRa sgRNAs vs. vector in E:T = 0.5:1, p < 0.0001. Error bars: All data points in this figure are presented as mean ± s.e.m. Asterisks: * p < 0.05, ** p < 0.01, *** p < 0.001. Additional supporting data:

Article Snippet: An AAV version of the CRISPR activation vector (AAV-CRISPRa vector, i.e. U6p-sgSapI-EF1a-MS2-p65-HSF1-sPA; Addgene, #129602), was generated by restriction cloning and Gibson assembly.

Techniques: Transduction, Plasmid Preparation, Flow Cytometry, Staining, Derivative Assay, Fluorescence, MANN-WHITNEY, Co-Culture Assay, Two Tailed Test, Cell Culture

Journal: Nature immunology

Article Title: Multiplexed activation of endogenous genes by CRISPRa elicits potent anti-tumor immunity

doi: 10.1038/s41590-019-0500-4

Figure Lengend Snippet: a, Schematics for generating genome-scale CRISPR activation (SAM) sgRNA library-transduced cells and assessment of tumorigenic capacity. b, Lentiviral CRISPRa-mediated transcriptional activation of Cd70 , Cd80 , Cd86 , Ifnα4 , Ifnβ1 , and Ifnγ , normalized to vector-transduced controls. n = 3 infection replicates from one representative experiment. Two-sided unpaired t-test, sgRNAs vs. vector Cd70 , p = 0.0006; Cd80 , p = 0.0025; Cd86 , p = 0.026; Ifnα4 , p = 0.0007; Ifnβ1 , p = 0.003; Ifnγ , p < 0.0001. c, Tumor growth curves of Vector (E0771-Vector, n = 8 mice) or SAM transduced E0771-dCas9-VP64-MPH cells (E0771-SAM; n = 50) transplanted in C57BL/6J mice. Two-way ANOVA: p < 0.0001. d, Tumor growth curves of E0771-SAM cells in syngeneic immunocompetent mice (C57BL/6J, n = 5 mice) and two strains of immunodeficient mice (Nude ( Foxn1 nu ), n = 4; Rag1 −/− , n = 5). Two-way ANOVA: C57BL/6J vs. Nude ( Foxn1 nu ), p < 0.0001; C57BL/6J vs. Rag1 −/− , p < 0.0001; Nude ( Foxn1 nu ) vs. Rag1 −/− , p < 0.0001. e, Tumor growth curves of E0771-SAM cells, with (n = 4 mice) or without T cell depletion (n = 11). Two-way ANOVA, untreated vs. α-CD4 + α-CD8 treated, p < 0.0001. f, Schematics of the experimental design for harnessing multiplexed endogenous gene activation as immunotherapy (MAEGI) in a cellular vaccine formulation. The E0771 cell-based MAEGI (c-MAEGI) was generated by treating E0771-SAM cells (E0771 transduced with dCas9-VP64, MS2-P65-HSF1, and a genome-wide CRISPRa library) with mitomycin. The mock cell-vector treatment (Cell-Vector) was generated in parallel. g, Growth curves of orthotopic E0771 transplants in mice pre-inoculated with PBS, mock Cell-Vector vaccine, or c-MAEGI at various time points prior to tumor transplantation (PBS, n = 11 mice; E0771-Vector at dpi = −7 (abbreviated @-d7, n = 5); c-MAEGI@-d14, (n = 5); c-MAEGI@-d7, (n = 6); c-MAEGI@-d3, (n = 6)). Two-way ANOVA: Cell-Vector@-d7 vs. PBS, p = 0.2077; c-MAEGI@-d3 vs. PBS or Vector@-d7, p < 0.0001; c-MAEGI@-d7 vs. PBS or Vector@-d7, p < 0.0001; c-MAEGI@-d14 vs. PBS or Vector@-d7, p < 0.0001. h, E0771 tumor-bearing mice were treated with PBS (n = 16 mice), Cell-Vector (n = 5) or c-MAEGI (n = 8) at indicated times (blue arrows). Two-way ANOVA: PBS vs. Vector, p = 0.2955; c-MAEGI vs. PBS, p < 0.0001; c-MAEGI vs. Vector, p = 0.0005. i, Tumor growth curves in E0771 tumor-bearing mice that were treated by PBS (n = 8 mice), c-MAEGI (n = 8), α-CTLA4 (n = 6), or c-MAEGI + α-CTLA4 (n = 4). Blue arrows, c-MAEGI treatment; yellow arrows, α-CTLA4 treatment. Two-way ANOVA: c-MAEGI vs. PBS, p = 0.0105; α-CTLA4 vs. PBS, p < 0.0001; c-MAEGI + α-CTLA4 vs. α-CTLA4 alone, p = 0.0005; c-MAEGI + α-CTLA4 vs. c-MAEGI, p < 0.0001. Error bars: All data points in this figure are presented as mean ± s.e.m. Asterisks: * p < 0.05, ** p < 0.01, *** p < 0.001. Additional supporting data:

Article Snippet: An AAV version of the CRISPR activation vector (AAV-CRISPRa vector, i.e. U6p-sgSapI-EF1a-MS2-p65-HSF1-sPA; Addgene, #129602), was generated by restriction cloning and Gibson assembly.

Techniques: CRISPR, Activation Assay, Plasmid Preparation, Infection, Generated, Transduction, Genome Wide, Transplantation Assay

Journal: Nature immunology

Article Title: Multiplexed activation of endogenous genes by CRISPRa elicits potent anti-tumor immunity

doi: 10.1038/s41590-019-0500-4

Figure Lengend Snippet: a, Schematics of the experimental design for intratumoral delivery of MAEGI in an AAV formulation. AAV-g-MAEGI was generated by cloning the genome-scale activation sgRNA SAM library into an AAV-CRISPRa vector, followed by pooled viral packaging into AAV9. b, Growth curves of orthotopic E0771-dCas9-VP64 tumor transplants in C57BL/6J mice treated with PBS, AAV-Vector, or AAV-g-MAEGI by intratumoral administration at indicated times (blue arrows) (PBS, n = 8 mice; AAV-Vector, n = 6; AAV-g-MAEGI, n = 6). Two-way ANOVA: PBS vs. AAV-Vector, p = 0.0013; AAV-g-MAEGI vs. PBS, p < 0.0001; AAV-g-MAEGI vs. AAV-Vector, p = 0.0303. c-d, Tumor-specific immune responses in tumor-bearing C57BL/6J mice, assessed by measuring the proportion of Ifn-γ-producing splenocytes with ELISPOT assays after stimulation with mitomycin-treated tumor cells. c, Representative images of spots obtained from four mice for each treatment group (PBS, AAV-Vector, and AAV-g-MAEGI). d, Quantification of ELISPOT assays from mice treated with PBS (n = 4 mice), AAV-Vector (n = 7) or AAV-g-MAEGI (n = 9). Two-sided unpaired t-test: AAV-Vector vs. PBS, p = 0.326; AAV-g-MAEGI vs. PBS, p = 0.0009; AAV-g-MAEGI vs. AAV-Vector, p = 0.0004. Results shown are from the aggregation of 2 independent experiments. e, Growth curves of orthotopic B16F10-dCas9-VP64 tumor transplants in C57BL/6J mice treated with PBS, AAV-Vector, or AAV-g-MAEGI at indicated times (blue arrows) (PBS, n = 6 mice; AAV-Vector, n = 5; AAV-g-MAEGI, n = 6). Two-way ANOVA: AAV-Vector vs. PBS, p < 0.0001; AAV-g-MAEGI vs. PBS, p < 0.0001; AAV-g-MAEGI vs. AAV-Vector, p < 0.0001. f, Growth curves of Pan02-dCas9-VP64 tumors in C57BL/6J mice treated with PBS (n = 4 mice), AAV-Vector (n = 5), or AAV-g-MAEGI (n = 5) at indicated times (blue arrows). Two-way ANOVA: AAV-Vector vs. PBS, p < 0.0001; AAV-g-MAEGI vs. PBS, p < 0.0001; AAV-g-MAEGI vs. AAV-Vector, p < 0.0001. Error bars: All data points in this figure are presented as mean ± s.e.m. Asterisks: * p < 0.05, ** p < 0.01, *** p < 0.001. Additional supporting data:

Article Snippet: An AAV version of the CRISPR activation vector (AAV-CRISPRa vector, i.e. U6p-sgSapI-EF1a-MS2-p65-HSF1-sPA; Addgene, #129602), was generated by restriction cloning and Gibson assembly.

Techniques: Generated, Clone Assay, Activation Assay, Plasmid Preparation, Enzyme-linked Immunospot

Journal: Nature immunology

Article Title: Multiplexed activation of endogenous genes by CRISPRa elicits potent anti-tumor immunity

doi: 10.1038/s41590-019-0500-4

Figure Lengend Snippet: a, Schematics of experimental design. Exome sequencing of cancer cells and normal tissues was performed to identify somatic mutations. A library of CRISPRa sgRNAs targeting the top mutant genes was synthesized and pool-cloned into the AAV-CRISPRa vector, then packaged into AAVs to generate AAV-p-MAEGI. b, Tumor growth curves of E0771-dCas9-VP64 syngeneic tumors in mice treated by PBS (n = 24 mice), AAV-Vector (n = 20), or AAV-p-MAEGI (n = 25) by intratumoral administration at indicated times (blue arrows). Two-way ANOVA: AAV-Vector vs. PBS, p < 0.0001; AAV-p-MAEGI vs. PBS, p < 0.0001; AAV-p-MAEGI vs. AAV-Vector, p < 0.0001. c, Response rates for each treatment, categorized by near-complete or complete response (nCR/CR), partial response (PR), or stable/progressive disease (SD/PD). d, Long-term spider growth plots of E0771-dCas9-VP64 tumors treated by PBS, AAV-Vector, or AAV-p-MAEGI from ( b) . Mice whose tumors had undergone complete response by AAV-p-MAEGI (n = 9) were subjected to tumor re-challenges (green arrows). No tumors grew with re-challenge. Treatment days are indicated by blue arrows. e, Schematic of experimental design for evaluating the induction of systemic anti-tumor immunity by AAV-p-MAEGI. 2*10 6 or 0.25*10 6 E0771-dCas9-VP64 tumor cells were respectively transplanted into the left or right flank of C57BL/6J mice to model local and distant tumors. AAV-p-MAEGI was administered only into the local tumors at the indicated times (blue arrows). f-g, Growth curves of E0771-dCas9-VP64 local ( f ) and distant ( g ) tumors in mice treated by AAV-Vector (n = 18), or AAV-p-MAEGI (n = 18). Inset, pie charts detailing the response rates for each treatment. ( f ) Two-way ANOVA: AAV-p-MAEGI vs. AAV-Vector, p < 0.0001. ( g ) Two-way ANOVA: AAV-p-MAEGI vs. AAV-Vector, p = 0.0003. h, Schematics of dual AAV CRISPRa system for MAEGI delivery into tumor-bearing mice. i, Growth curves of E0771 syngeneic tumors in mice treated by intratumoral injection of PBS (n = 10 mice), AAV-dCas9 + AAV-Vector (n = 10), or dual AAV-p-MAEGI (n = 10) at the indicated time points (blue arrows). Two-way ANOVA: AAV-dCas9+AAV-Vector vs. PBS, p = 0.0083; dual AAV-p-MAEGI vs. PBS, p < 0.0001; dual AAV-p-MAEGI vs. AAV-dCas9+AAV-Vector, p = 0.0002. Error bars: All data points in this figure are presented as mean ± s.e.m. Asterisks: * p < 0.05, ** p < 0.01, *** p < 0.001. Additional supporting data: , , and

Article Snippet: An AAV version of the CRISPR activation vector (AAV-CRISPRa vector, i.e. U6p-sgSapI-EF1a-MS2-p65-HSF1-sPA; Addgene, #129602), was generated by restriction cloning and Gibson assembly.

Techniques: Sequencing, Mutagenesis, Synthesized, Clone Assay, Plasmid Preparation, Injection

Journal: Cancer cell

Article Title: A transcription factor addiction in leukemia imposed by the MLL promoter sequence

doi: 10.1016/j.ccell.2018.10.015

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The previous published three-vector CRISPR SAM system (dCAS-VP64_Blast, Addgene:61425; MS2-P65-HSF1_Hygro, Addgene: 61426; sgRNA(MS2)_zeo, Addgene:61427) was adapted for activation of the endogenous MLL gene ( Konermann et al., 2015 ).

Techniques: Recombinant, SYBR Green Assay, Gel Extraction, Purification, Sample Prep, Clone Assay, Luciferase, Sequencing, Software

Journal: Cancer cell

Article Title: A transcription factor addiction in leukemia imposed by the MLL promoter sequence

doi: 10.1016/j.ccell.2018.10.015

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The previous published three-vector CRISPR SAM system (dCAS-VP64_Blast, Addgene:61425; MS2-P65-HSF1_Hygro, Addgene: 61426; sgRNA(MS2)_zeo, Addgene:61427) was adapted for activation of the endogenous MLL gene ( Konermann et al., 2015 ).

Techniques: Recombinant, SYBR Green Assay, Gel Extraction, Purification, Sample Prep, Clone Assay, Luciferase, Sequencing, Software

Journal: bioRxiv

Article Title: Genome and epigenome engineering CRISPR toolkit for probing in vivo cis -regulatory interactions in the chicken embryo

doi: 10.1101/135525

Figure Lengend Snippet: (A) Schematic of dCas9-VP64-MCP SAM complex. (B) One sgRNA targeting the Sox10 promoter co-electroporated with dCas9-VP64 and MCP-VP64 causes a premature activation of Sox10 transcription on the experimental (left, magenta) as compared to the control side (right, blue), 50% embryos, n=12, *p<0.05 ). Results confirmed in three independent experiments; representative embryos are shown, error bars represent the standard deviation.

Article Snippet: The MCP-VP64 construct was generated by removing the IRES-H2B-RFP cassette from pCI_H2B-RFP vector ( ) and inserting the MCP and VP64 fragments (amplified from Addgene #61423 and #47319, respectively) using 2-fragment Infusion cloning.

Techniques: Activation Assay, Control, Standard Deviation