electroporation  (Bio-Rad)

Journal: bioRxiv

Article Title: Next-generation all-in-one CRISPR/Cas9 multiply-edited CD30CAR-T cells: Potency despite risk of translocations

doi: 10.64898/2026.02.27.708432

Figure Lengend Snippet: (A) Scheme of the homology-directed repair template (HDRT) encoding the CAR and promoting specific integration into the T cell receptor alpha constant (TRAC) locus with TRAC homology arms (HA), transmembrane and endocytoplasmic CD28 domains, and CD3zeta signaling domain. The sequences for scFvs comprised a mouse anti-CD19, a mouse anti-CD30 (HRS-3) and human anti-CD30 sequences (5F11, 2H9). The detection domains included IgG4 hinge with IgG1-CH3 (FcIg). (B) Scheme of CAR-T cell production. After gradient separation, peripheral blood mononuclear cells (PBMCs) were activated for 2-5 days with anti-CD3 and anti-CD28 activating beads in the presence of IL-7 and IL-15. Prior to electroporation on day 0, the cells were mixed with gRNA, Cas9, HDRT, PGA, and an enhancer. After electroporation, cells were maintained in culture in the presence of IL-7 and IL-15 and harvested at different time points for analyses. (C) Flow cytometry analysis of CAR expression on 7 post electroporation for comparison of the different HDRTs/ scFvs, via detection of the CAR through the IgFc domain (representative data, n=1 donor). Not electroporated cells and cells electroporated with RNPs but no HDRT ( TRAC KO ) are shown as controls. Lack of CD3 detection indicates TCR knockout. (D) Flow cytometry analysis of CAR expression on days 2, 7, and 9 post electroporation as quantified data comparing the expression of the CARs incorporating different scFvs (CD19CAR in grey, HRS-3-CD30CAR in green, and 5F11-CD30CAR in blue) and detected via the FcIg domain (n=3 donors). (E) Flow cytometry analysis of CAR expression on day 9 post electroporation separated for CD4 + and CD8 + cells as quantified data comparing the expression of the CARs incorporating different scFvs (CD19CAR in grey, HRS-3-CD30CAR in green, and 5F11-CD30CAR in blue) (n=3 donors). Statistical significance is annotated: n.s.: not significant, *: p>0.05.

Article Snippet: An ExPERT ATx TM Electroporation® device (MaxCyte) was used for electroporation with the “Resting T cell 14-3” program.

Techniques: Electroporation, Flow Cytometry, Expressing, Comparison, Knock-Out

Journal: bioRxiv

Article Title: Next-generation all-in-one CRISPR/Cas9 multiply-edited CD30CAR-T cells: Potency despite risk of translocations

doi: 10.64898/2026.02.27.708432

Figure Lengend Snippet: (A) Schematic overview of HDRT incorporating the tCD34 hinge for 5F11-CD30CAR-T cell generation. (B) Analysis of CAR expression. Representative flow cytometry analyses of CAR expression on day 12 after electroporation. TRAC KO cells, electroporated RNPs but no HDRT, are shown as controls. Lack of CD3 detection indicates TCR knock-out. (C) Percentages and absolute numbers of CD4 + and CD8 + CD30CAR-T cells detectable on day 5 and day 12 after electroporation showing expansion (n=3 donors). (D) Immunophenotypes of CD30CAR-T cells defined by expression of CD62L and CD45RA. Representative flow cytometric analysis of phenotypes of CD8 + and CD4 + CAR-T cells on day 12 after electroporation. N: Naïve, CM: central memory, EM: effector memory, TE: Terminal effector. (E) Quantified frequencies of naïve (N) and central memory (CM) phenotypes within CD8 + and CD4 + CD30CAR-T cells on day 5 and 12 after electroporation indicating their accumulation upon culture (n=3 donors). (F) Effectors (TRAC KO hatched lines and CAR KI blue lines) were cultured for 72 h with targets L540/fluc-GFP (EBV-negative) and L591/fluc-GFP (EBV-positive). Assay was performed with different Effector : Target ratios (0.03:1, 0.01:1, 0.3:1, 1:1, 3:1, 10:1) (n=3 donors). Cytotoxicity was calculated based on bio-luminescence remaining in target cells. Statistical significance is annotated: n.s.: not significant, *: p>0.05.

Article Snippet: An ExPERT ATx TM Electroporation® device (MaxCyte) was used for electroporation with the “Resting T cell 14-3” program.

Techniques: Expressing, Flow Cytometry, Electroporation, Knock-Out, Cell Culture

Journal: bioRxiv

Article Title: Next-generation all-in-one CRISPR/Cas9 multiply-edited CD30CAR-T cells: Potency despite risk of translocations

doi: 10.64898/2026.02.27.708432

Figure Lengend Snippet: (A) Flow cytometry analyses of CAR expression within CAR KI -T and CAR KI PD-1 K -T cells on day 12 after electroporation. Representative flow cytometry plots showing CD8 + and CD4 + CD30CAR-T cells. (B) CD8 + and CD4 + CD30CAR-T cells co-electroporated with gRNA 1 showed efficient PD-1 KO . Representative flow cytometry analyses. (C) Quantitative analyses showing expansion of TRAC KI or additional PD-1 KO CD30CAR-T cells separated between CD8 + and CD4 + populations at days 5 and 12 after electroporation (n=3 donors). (D) Quantitative analyses showing naïve or central memory phenotypes for TRAC KI or additional PD-1 KO CD30CAR-T cells separated between CD8 + and CD4 + populations from day 5 to 12 after electroporation (n=3 donors). Statistical significance is annotated: n.s.: not significant, *: p>0.05, **: p>0.005.

Article Snippet: An ExPERT ATx TM Electroporation® device (MaxCyte) was used for electroporation with the “Resting T cell 14-3” program.

Techniques: Flow Cytometry, Expressing, Electroporation

Journal: bioRxiv

Article Title: Next-generation all-in-one CRISPR/Cas9 multiply-edited CD30CAR-T cells: Potency despite risk of translocations

doi: 10.64898/2026.02.27.708432

Figure Lengend Snippet: (A) Representative flow cytometry analyses of CAR KI , CAR KI HLA-I KO and CAR KI HLA-I KO PD-1 KO T cells on day 12 after electroporation, showing CD8 + and CD4 + CD30CAR-T cells. (B) CD8 + and CD4 + CD30CAR-T cells co-electroporated with gRNAs for simultaneous HLA-I KO and PD-1 KO . Representative flow cytometry analyses. (C) Quantitative analyses showing expansion of CD8 + and CD4 + CD30CAR-T cells with CAR KI , CAR KI HLA-I KO or CAR KI HLA-I KO PD-1 KO at days 5 (left panels) and 12 8right panels) after electroporation (n=3 donors). Statistical significance is annotated: n.s.: not significant, *: p>0.05, **: p>0.005.

Article Snippet: An ExPERT ATx TM Electroporation® device (MaxCyte) was used for electroporation with the “Resting T cell 14-3” program.

Techniques: Flow Cytometry, Electroporation

Journal: bioRxiv

Article Title: Next-generation all-in-one CRISPR/Cas9 multiply-edited CD30CAR-T cells: Potency despite risk of translocations

doi: 10.64898/2026.02.27.708432

Figure Lengend Snippet: (A) Effector cells were generated with 3 donors (A, B, C), and were expanded for 12 days after electroporation. The cells were cryopreserved, thawed and then co-cultured with L540, L591 or Myla cells co-expressing fluc-GFP. Effector : Target (E:T) ratios 3:1, 1:1, 0.6:1, 0.3:1 and 0.1:1 were set up and cytotoxicity was detected by bio-luminescence analyses after 72 h of co-culture. (B) Cytotoxicity at an Effector : Target ratio of 1:1. Data were merged from 3 different donors. Statistical analyses showed no significant differences among arms.

Article Snippet: An ExPERT ATx TM Electroporation® device (MaxCyte) was used for electroporation with the “Resting T cell 14-3” program.

Techniques: Generated, Electroporation, Cell Culture, Expressing, Co-Culture Assay

Journal: bioRxiv

Article Title: Next-generation all-in-one CRISPR/Cas9 multiply-edited CD30CAR-T cells: Potency despite risk of translocations

doi: 10.64898/2026.02.27.708432

Figure Lengend Snippet: Chromosomal rearrangements were evaluated by CAST-Seq on day 14 after electroporation employing at least two CAST-Seq libraries. Shown are chromosomal translocations with (A) TRAC and (B) PDCD1 target sites. Green line, ON-target aberrations; magenta, chromosomal translocations between target sites; red lines, off-target activity-mediated translocations. (C) Indel frequencies as determined by targeted amplicon sequencing of ON-target and OFF-target sites nominated by CAST-Seq.

Article Snippet: An ExPERT ATx TM Electroporation® device (MaxCyte) was used for electroporation with the “Resting T cell 14-3” program.

Techniques: Electroporation, Activity Assay, Amplification, Sequencing