Journal: bioRxiv
Article Title: Ultra-large targeted DNA integrations in primary human cells
doi: 10.64898/2026.04.09.717505
Figure Lengend Snippet: (a) Timeline of T cell knock-in electroporation workflow, knock-in strategy and designs for BCMA-CAR (1.6 kb integration) across the series of DNA HDR template formats tested. (b) Comparison of BCMA-CAR HDRTs at concentrations 5nM-160nM or 0-100E3 MOI in terms of knock-in efficiency, (c) live cell count per 1e6 edited cells, and (d) knock-in cell count per 1e6 edited cells measured 7 days post electroporation. (e) Knock-in of a logic-gated synNotch circuit (5.6 kb integration) using linear ssDNA + tCTS, circular cssDNA + CTS, and nanoplasmid + CTS templates with corresponding knock-in efficiency, live cell count and knock-in cell count 7 days post electroporation using Cas9 mRNA. Circular cssDNA was produced and provided by Kano Therapeutics. (f) Knock-in strategy and designs for a logic-gated synNotch circuit (5.6 kb integration) at concentrations 5nM-160nM with corresponding knock-in efficiency, live cell count, and knock-in cell count 7 days post electroporation using Cas9 RNP or Cas9 mRNA. Each experiment was performed with T cells from two independent healthy human blood donors represented by individual dots plus mean. CTS, Cas9 target site. RNP, ribonucleoprotein. MOI, multiplicity of infection.
Article Snippet: To stain for BCMA-CAR, cells were incubated with 0.3 μg BCMA recombinant protein conjugated to biotin (Biotinylated Human BCMA / TNFRSF17 Protein, His, Avitag, AcroBioSystems, BCA-H82E4_200ug) for 15 minutes at room temperature prior to surface cell staining.
Techniques: Knock-In, Electroporation, Comparison, Cell Characterization, Produced, Infection