trac-specific guide rna  (GenScript corporation)

Journal: bioRxiv

Article Title: Genome editing human primary T cells with microfluidic vortex shedding & CRISPR Cas9

doi: 10.1101/2020.02.26.960336

Figure Lengend Snippet: Cas9 and locus-specific TRAC-1 gRNAs (as Cas9-RNP complexes) were delivered to activated primary human CD3+ T cells via μVS or electroporation. Quantification of A , percentage of TCR knock-out cells, B , total viable, TCR knock-out cells, C , percentage of viable T cells and D , total viable T cells were performed at Days 1, 4, 7, 10 and 14 post-transfection. TCR expression was measured via CD3 and TCRa/b co-staining. TCR knockout was quantified as a percentage of CD3- and TCRa/b-double negative cells and the total number of live cells. Propidium iodide exclusion gating and event collection rate were used to measure viability and cell concentration, respectively.. Non-targeting gRNA as a Cas9-RNP complex served as a non-editing control. Non-transfected samples served as a negative control. Data represents mean ± SD of biological triplicate. P-values by unpaired, two-tailed, heteroscedastic T-tests.

Article Snippet: Cas9 (Invitrogen) and TRAC-1-specific single-guide RNAs (sgRNAs, Synthego), as a Cas9-RNP complex (1:1 Cas9:gRNA molar ratio) were delivered primary human CD3+ T cells after 2 days of Anti-CD3/-CD28 Dynabead (ThermoFisher) stimulation.

Techniques: Electroporation, Knock-Out, Transfection, Expressing, Staining, Concentration Assay, Negative Control, Two Tailed Test

Journal: bioRxiv

Article Title: Genome editing human primary T cells with microfluidic vortex shedding & CRISPR Cas9

doi: 10.1101/2020.02.26.960336

Figure Lengend Snippet: A , CD25, and B, PD-1 expression levels were quantified in cells transfected with μVS or electroporation. Surface marker expression levels were measured in the total live cell and TRAC-1 KO live population via flow cytometry at Days 1, 4, 7, 10 and 14 post-transfection. C , ELISA quantification of IFNg supernatant levels in transfected and non-transfected cells at Day 1, 4, 7, and 10 post-transfection. Non-targeting gRNA as a Cas9-RNP complex served as a non-editing control. Cells mixed with P3 buffer or Opti-MEM buffer media that were not transfected served as non-transfection controls for electroporation and μVS samples, respectively. Data represent mean ± SD of 3 wells. P-values by unpaired, two-tailed, heteroscedastic T-tests.

Article Snippet: Cas9 (Invitrogen) and TRAC-1-specific single-guide RNAs (sgRNAs, Synthego), as a Cas9-RNP complex (1:1 Cas9:gRNA molar ratio) were delivered primary human CD3+ T cells after 2 days of Anti-CD3/-CD28 Dynabead (ThermoFisher) stimulation.

Techniques: Expressing, Transfection, Electroporation, Marker, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Genome editing human primary T cells with microfluidic vortex shedding & CRISPR Cas9

doi: 10.1101/2020.02.26.960336

Figure Lengend Snippet: TRAC-1-targeting Cas9-RNPs were delivered to activated primary human CD3+ T cells via eμVS with an applied electrical field. Quantification of A , percentages of TCR knock-out cells and B , viable T cells were quantified via flow cytometry at multiple timepoints post-transfection. TCR knockout levels were measured via TCRa/b staining and quantified as a percentage of TCRa/b-negative cells and the total number of live cells. eμVS with no applied electric field (0 kV cm-1) and non-transfection samples served as controls. Data represent the means ± SD of n ≥ 2 samples per condition. Statistical analysis performed between groups indicated on graphs. Dot plots representative of replicate samples. P<0.05 by unpaired, two-tailed, heteroscedastic T-tests.

Article Snippet: Cas9 (Invitrogen) and TRAC-1-specific single-guide RNAs (sgRNAs, Synthego), as a Cas9-RNP complex (1:1 Cas9:gRNA molar ratio) were delivered primary human CD3+ T cells after 2 days of Anti-CD3/-CD28 Dynabead (ThermoFisher) stimulation.

Techniques: Knock-Out, Flow Cytometry, Transfection, Staining, Two Tailed Test