Journal: Nature

Article Title: Reprogramming human T cell function and specificity with non-viral genome targeting

doi: 10.1038/s41586-018-0326-5

Figure Lengend Snippet: a, Except where explicitly noted otherwise, we use viability to refer to the number of live cells in an experimental condition (expressed as a %) relative to an equivalent population that went through all protocol steps except for the actual electroporation (No electroporation control). We use the term efficiency to refer to the percentage of live cells in a culture expressing the “knocked in” exogenous sequence (such as GFP). Finally, the total number of cells positive for the desired modification was calculated by multiplying the efficiency by the absolute cell count. Methodological changes that maximized efficiency were not always optimal for the total number of positive cells, and vice-versa. b, Double-stranded (ds)DNA, both circular (plasmid) and linear, when electroporated into primary human T cells, caused marked loss in viability with increasing amounts of template. Co-delivery of an RNP caused less reduction in viability post electroporation. Of note, in these experiments no loss in viability was seen with short single-stranded (ss)DNA oligo donor nucleotides (ssODNs). c, RNPs must be delivered concurrently with DNA to see increased viability. T cells from two donors were each electroporated twice with an eight-hour rest in between electroporations. While two electroporations so closely interspersed caused a high degree of cell death, delivery of the RNP and linear dsDNA template could be delivered separately. Initial RNP electroporation did not protect from the loss of viability if dsDNA was delivered alone in the second round of electroporation. d, We determined whether the order of adding reagents influenced targeting efficiency and viability. In wells where the RNP and the DNA HDR template were mixed together prior to adding the cells (1. RNP + HDRT; 2. + Cells), there was a marked increase in targeting efficiency. e, Note, with the high concentration of dsDNA used in these experiments, viability was higher if the RNP and cells were mixed first and the DNA template was added immediately prior to electroporation (1. RNP + Cells; 2. + HDRT). Taken together, these data likely suggest that pre-incubation of the RNP and HDR template, even for a short period, increased the amount of DNA HDR template delivered into the cell, which increased efficiency but decreased viability. However, viability after RNP and dsDNA HDR template pre-incubation was still higher than was observed with dsDNA HDR template electroporation by itself ( b ). 5 μg of dsDNA HDR temple was used in ( c-e ). f, Primary human T cells were cultured for 2 days using varying combinations of anti-CD3/CD28 T cell receptor (TCR) stimulation and cytokines prior to electroporation of RAB11A targeting RNP and HDR template, followed by varying culture conditions post-electroporation. g, Among the RNP and HDR template concentrations tested here, optimal GFP insertion into RAB11A was achieved at intermediate concentrations of the RNP and dsDNA HDRT. h, Arrayed testing of electroporation pulse conditions showed that, in general, conditions yielding higher HDR efficiency decreased viability. EH115 was selected to optimize efficiency, while still maintaining sufficient viability. i, Diagrammatic timeline of non-viral genome targeting. Approximately one week is required to design, order from commercial suppliers, and assemble any novel combination of genomic editing reagents (gRNA and the HDR template). Two days prior to electroporation, primary human T cells isolated from blood or other sources are stimulated. dsDNA HDR templates can be made easily by PCR followed by a SPRI purification to achieve a highly concentrated and pure product suitable for electroporation. On the day of electroporation, the gRNA (complexed with Cas9 to form an RNP), the HDR template, and harvested stimulated T cells are mixed and electroporated, a process taking approximately 1.5 hours. After electroporation, engineered T cells can be readily expanded for an additional 1-2 weeks. Viability was measured 2 days following electroporation and GFP expression was measured at day 4. Graphs display mean ( b, c, g, h ) and/or individual donor values ( b-h ) in n=2 independent healthy donors ( b-h ). For d, e, and h one representative donor is shown.

Article Snippet: Immediately prior to electroporation, de-beaded cells were centrifuged for 10 minutes at 90g, aspirated, and resuspended in the Lonza electroporation buffer P3 using 20 μL buffer per one million cells.

Techniques: Electroporation, Expressing, Sequencing, Modification, Cell Counting, Plasmid Preparation, Concentration Assay, Incubation, Cell Culture, Isolation, Purification

Journal: Nature

Article Title: Reprogramming human T cell function and specificity with non-viral genome targeting

doi: 10.1038/s41586-018-0326-5

Figure Lengend Snippet: a, HDR mediated integration of a GFP fusion tag to the housekeeping gene Rab11A. b, Development and optimization of non-viral genome targeting for both cell viability and HDR efficiency. c, Insertion of a GFP fusion into the endogenous RAB11A gene using non-viral targeting in primary human CD4+ and CD8+ T cells. d, Average efficiency with the RAB11A-GFP HDR template was 33.7% and 40.3% in CD4+ and CD8+ cells respectively. e, Viability (number of live cells relative to non-electroporated control) after non-viral genome targeting averaged 68.6%. Efficiency and viability were measured 4 days following electroporation. Mean of n=12 independent healthy donors displayed ( d-e ). See also .

Article Snippet: Immediately prior to electroporation, de-beaded cells were centrifuged for 10 minutes at 90g, aspirated, and resuspended in the Lonza electroporation buffer P3 using 20 μL buffer per one million cells.

Techniques: Electroporation

Journal: Nature

Article Title: Reprogramming human T cell function and specificity with non-viral genome targeting

doi: 10.1038/s41586-018-0326-5

Figure Lengend Snippet: a, Diagram of in vivo human antigen specific tumour xenograft model. 8 to 12 week old NSG mice were seeded with 1×10 6 A375 cells (human melanoma cell line; NY-ESO-1 antigen+ and HLA-A*0201+) subcutaneously in a shaved flank. Primary human T cells edited to express an NY-ESO-1 antigen specific TCR were generated (either through lentiviral transduction or non-viral TCR replacement), expanded for 10 days following transduction or electroporation, and frozen. Either a bulk edited population was used ( b,c ) or a NY-ESO-1 TCR+ sorted population ( d-f ) was used. At seven days post tumour seeding, T cells were thawed and adoptively transferred via retro-orbital injection. b, Two days following transfer of 5×10 6 bulk non-virally targeted T cells (~10% TCR+ NYESO-1+ (Red), ~10% TCR+ NYESO-1- (Orange), and ~80% TCR- NYESO-1- (Green), see ), NY-ESO-1+ non-virally edited T cells preferentially accumulated in the tumour vs. the spleen. n=5 mice for each of four human T cell donors. c, Ten days following transfer of 5×10 6 bulk non-virally targeted CFSE labeled T cells, NYESO-1 TCR+ cells showed greater proliferation than TCR- or TCR+NYESO-1- T cells, and showed greater proliferation (CFSE Low) in the tumour than in the spleen. At ten days post transfer TCR- and TCR+NYESO- T cells were difficult to find in the tumour . d, Individual longitudinal tumour volume tracks for data summarized in . 3×10 6 sorted NY-ESO-1 TCR+ T cells generated either by lentiviral transduction (Black) or non-viral TCR replacement (Red) were transferred on day 7 post tumour seeding and compared to vehicle only injections until 24 days post tumour seeding. Note that the same data for vehicle control data are shown for each donor in comparison to lentiviral delivery (above) and non-viral TCR replacement (below). e,f, In these experiments, seventeen days following T cell transfer ( d ), non-virally TCR replaced cells appeared to show greater NY-ESO-1 TCR expression and lower expression of exhaustion markers. Transfer of both lentivirally transduced and non-viral TCR replaced cells showed significant reductions in tumour burden on day 24. In this experimental model, non-viral TCR replacement showed further reductions compared to the lentiviral transduction , potentially due to knockout of the endogenous TCR, endogenous regulation of the new TCR’s expression, some difference in the cell populations amenable to non-viral vs lentiviral editing, or confounding variables in cell handling between lentiviral transduction and non-viral genome targeting. n=4 ( b ), n=2 ( d-f ), or n=1 ( c ) independent healthy donors in 5 ( b, c ) or 7 mice ( d-f ) per donor with mean ( b, e, f ) and standard deviation ( b ).

Article Snippet: Immediately prior to electroporation, de-beaded cells were centrifuged for 10 minutes at 90g, aspirated, and resuspended in the Lonza electroporation buffer P3 using 20 μL buffer per one million cells.

Techniques: In Vivo, Generated, Transduction, Electroporation, Injection, Labeling, Expressing, Knock-Out, Standard Deviation

Journal: eLife

Article Title: A functional genetic toolbox for human tissue-derived organoids

doi: 10.7554/eLife.67886

Figure Lengend Snippet: ( a ) Efficiency quantification of different DNA delivery methods. EGFP-positive cells were quantified by flow cytometry 72 hr after transfection or transduction. N = 4 (or 3 for nucleofection EA104) different organoid lines were used for each condition. Error bars are plotted to show mean ± SEM. ( b ) Representative images showing DNA delivery efficiencies of different methods 72 hr after transfection/transduction. Top panel: widefield images; bottom panel: GFP channel. Scale bar denotes 100 μm. ( c ) T7 endonuclease assay showing the DNA cleavage efficiency of different CRISPR methods on the ACTB locus. Arrows denote lower bands generated by T7 endonuclease cutting. Left panel: schematic showing the different methods tested for introducing the Cas9 complex. cr/tr RNP, synthetic crispr/tracer RNA heterodimer with Cas9 RNP; ssRNP, single-strand synthetic guide RNA with Cas9 RNP. ( d ) Quantification of indels produced by the different CRISPR methods tested using ICE online analysis software ( https://ice.synthego.com/ ). N = 3 different organoid lines were used for each condition. Error bars are plotted to show mean ± SEM.

Article Snippet: 6 × 10 5 cells were suspended using Lonza Nucleofection P3 buffer, mixed with 6 μg of SOX9 reporter repair template plasmid.

Techniques: Flow Cytometry, Transfection, Transduction, CRISPR, Generated, Produced, Software

Journal: eLife

Article Title: A functional genetic toolbox for human tissue-derived organoids

doi: 10.7554/eLife.67886

Figure Lengend Snippet: ( a ) Schematic of the Organoid Easytag workflow. ssRNP and a circular plasmid repair template are nucleofected into dissociated cells at day 0. By day 3, cells have proliferated to become tiny colonies and are removed from the Matrigel and dissociated for selection by flow cytometry. EGFP + cells are re-plated sparsely (~1000–1500 cells/well of a 24-well plate) and grown until day 15 when organoids reach a sufficient size to be manually picked under a fluorescent microscope. Typically, 10–40 organoid colonies formed per ~1000 cells seeded. Organoids are picked into individual wells and passaged until sufficient cells are obtained for both genotyping and freezing down the line. Cells with red nuclei represent incorrectly targeted cells. Cells with white nuclei denote correctly targeted cells. ( b ) Schematic of repair template design for N terminal fusion mEGFP-ACTB gene targeting and the final product. Arrow shows the position of gRNA. E1, exon 1; E2, exon 2; 5′HA, 5′ homology arm; 3′HA, 3′ homology arm. ( c ) Representative flow cytometry results showing the percentage of EGFP cells 72 hr after nucleofection is performed. ( d ) Representative image showing mEGFP-ACTB organoid. DIC channel on the left and EGFP channel on the right. ( e ) Immunofluorescence of mEGFP-ACTB organoids. Blue: DAPI (nuclei); green: EGFP (ACTB fusion protein); red: SOX9 (lung progenitor marker). ( f ) Schematic of the AAVS1 targeting repair template design and the final product. E1, exon 1, E2, exon 2. Arrow indicates the position of the gRNA. ( g ) Immunofluorescence of AAVS1-EF1a-mTagRFP-T organoids. Blue: DAPI (nuclei); red: mTagRFP-T (membrane localised reporter); white: SOX2 (lung progenitor marker). Scale bars in all panels denote 50 μm.

Article Snippet: 6 × 10 5 cells were suspended using Lonza Nucleofection P3 buffer, mixed with 6 μg of SOX9 reporter repair template plasmid.

Techniques: Plasmid Preparation, Selection, Flow Cytometry, Microscopy, Immunofluorescence, Marker

Journal: eLife

Article Title: A functional genetic toolbox for human tissue-derived organoids

doi: 10.7554/eLife.67886

Figure Lengend Snippet: ( a ) Workflow for testing different homology-directed repair (HDR)-enhancing drugs. Organoid cells were treated with DMSO, RS-1, L755507 and SCR-7 for 48 hr after nucleofection for mEGFP-ACTB gene targeting. The percentage of EGFP-positive cells was analysed 72 hr after nucleofection. DMSO treatment was used as a negative control. ( b ) Summary of percentage of EGFP positive. No significant improvement of targeting efficiency was observed after the drug treatments. N = 3 different organoid lines were used. Error bars are plotted to show mean ± SEM.

Article Snippet: 6 × 10 5 cells were suspended using Lonza Nucleofection P3 buffer, mixed with 6 μg of SOX9 reporter repair template plasmid.

Techniques: Negative Control

Journal: Cell Genomics

Article Title: SpRY-mediated screens facilitate functional dissection of non-coding sequences at single-base resolution

doi: 10.1016/j.xgen.2024.100583

Figure Lengend Snippet:

Article Snippet: P3 Lonza buffer with supplement , Lonza , Cat#: V4XP-3032.

Techniques: Sequencing, Chromatin Immunoprecipitation, Binding Assay, Software, Recombinant, Modification, SYBR Green Assay, Purification, Sensitive Assay, Protease Inhibitor, Luciferase

Journal: The EMBO Journal

Article Title: CARD8 inflammasome activation triggers pyroptosis in human T cells

doi: 10.15252/embj.2020105071

Figure Lengend Snippet:

Article Snippet: P3 Primary Cell 96‐well Kit , Lonza , #V4SP‐3096.

Techniques: Sequencing, Synthesized, Enzyme-linked Immunosorbent Assay, Red Blood Cell Lysis, Cell Isolation, Sterility, Recombinant, CyQUANT Assay, LDH Cytotoxicity Assay, Staining, Bicinchoninic Acid Protein Assay, Protease Inhibitor, Western Blot, Membrane, Software