Application Note

CRISPR-Cas9–Mediated Knock-out of T Cell Receptor in Primary Human T Cells

Updated — July 17, 2026

Abstract

Traditional electroporation systems suffer from several difficulties, including (1) tedious optimization, (2) inflexibility, and (3) poor scaling from R&D to clinical volumes. Here, we use CyteQuest's electroporation platform for highly efficient CRISPR/Cas9-mediated gene knockout in primary T cells, demonstrating:

  1. Efficient, automated screening of voltage waveforms to optimize transfection
  2. Flexibility in voltage waveform and processing volume
  3. Identical transfection performance when scaling between cell processing speeds

Experimental workflow

1 day 2 days 3 days DAY −1 DAY 0 DAY 2 DAY 5 Thaw Primary human T cells Activate TransAct (Miltenyi) Transfect Cas9 RNP · CytoZap Scan Measure TCR knockout % · flow
Experimental workflow. Primary human T cells were thawed (Day −1), activated with TransAct (Miltenyi) (Day 0), and electroporated with a CRISPR-Cas9 RNP targeting the T cell receptor on the CytoZap Scan (Day 2). T cell receptor knockout was quantified by flow cytometry (Day 5).

Results

Programmable bipolar rectangular waveforms

Each well was electroporated with a bipolar rectangular waveform defined by three parameters: voltage amplitude (V), pulse duration (t), and frequency (f). Sweeping these parameters across the plate lets the CytoZap Scan identify the combination that best balances TCR knockout (KO), viability, and recovery.

Schematic of a bipolar rectangular waveform: a positive pulse of amplitude V and duration t, followed by an inverted pulse of equal magnitude at the half-period, repeating at frequency f.
Figure 1. Schematic of the bipolar rectangular waveform applied during screening. A pulse of amplitude V and duration t is followed by an inverted pulse of equal magnitude and duration at the half-period; the pattern repeats at frequency f. Schematic, not to scale.

Automated waveform optimization of T cell receptor knock-out in 24-well plate using CytoZap Scan

Activated T cells were screened with varying electrical waveforms using the CytoZap Scan to optimize TCR KO percentage, cell viability, and cell recovery. Optimization was performed rapidly (<2 minutes) using ~70 µL of cells per well, identifying a range of effective waveform parameters.

24-well plate map summarizing a combined optimization score (TCR knock-out × viability × recovery) across bipolar rectangular waveforms screened on the CytoZap Scan; each screened well is annotated with its voltage, frequency, and pulse duration. A1/A4 are transition conditions and A2/A3 are zero-voltage controls; well A5 scores highest at 68%.
Figure 2. Activated T cells were mixed with 1 µM CRISPR-Cas9 ribonucleoproteins (RNPs) targeting T cell receptor (TCR) and 2 µM electroporation enhancer (IDT Cat. #1075916) and transfected with varying electrical waveforms using the CytoZap Scan. Cell recovery was measured 24-h post-transfection and defined as the number of cells in each well normalized to the number of zero-voltage control cells. Cell viability was measured 24-h post-transfection using Trypan blue. TCR expression was measured 72-h post-transfection using surface staining and flow cytometry.
View raw data
72 h24 h24 h
WellVoltage (V)Duration (µs)Frequency (Hz)Avg current (mA)TCR exp. (%)ViabilityRecovery
A20———90%95%95%
A30———87%95%105%
A512100221802.5%95%73%
A613100221970.91%90%61%
B614100222140.32%88%57%
B51263221798.8%95%75%
B41663222491.5%90%59%
B32063223180.11%78%41%
B21463112105.8%92%65%
B11863112801.7%91%51%
C12263113500.32%86%38%

A2 and A3 are zero-voltage controls. Well A5 (highlighted) gave the best balance of knockout, viability, and recovery.

Screening identified multiple waveforms with >95% TCR knockout with varying impacts to cell viability and cell recovery. The best balance between editing efficiency and cell health was identified as the waveform in A5 which yielded 97.5% TCR knockout, 95% cell viability, and 73% cell recovery.

TCR expression by flow cytometry, two wells side by side. Left: control well A2, 89.1% TCR expression. Right: electroporated well A5, 2.5% TCR expression, indicating >97% knockout.
Figure 3. Control (Well A2) and electroporated (Well A5) T cells were analyzed by flow cytometry 3 days after electroporation to measure TCR expression.

Scaling

Optimized on the CytoZap Scan, transferred to the CytoZap Max

For this scaling demonstration, the waveform in well A6 was selected and transferred from the CytoZap Scan to the CytoZap Max, our large-volume system. This waveform was selected to balance cell health while yielding >98% TCR KO. T cells were electroporated in either the Scan or the Max and transfection performance was measured as described in Figure 2. TCR expression, viability, and cell recovery were 1–2% different between both platforms, confirming a waveform optimized on the Scan transfers seamlessly to the Max without re-optimization.

CytoZap ScanR&D screening CytoZap MaxClinical scale
TCR expression1.8%0.82%
Cell viability91%92%
Cell recovery59%61%

Values are from a paired, same-day run on each instrument. TCR expression is residual — lower values indicate greater knockout.

Summary

Key findings

  • Fully programmable waveforms. Voltage amplitude, pulse duration, and frequency are all tunable to the cell and cargo for balancing transfection efficiency and cell health.
  • Automated optimization. The CytoZap Scan screens waveforms across a 24-well plate to rapidly optimize TCR knockout, cell viability, and cell recovery.
  • Near-complete knockout, healthy cells. Optimized waveforms could achieve >99% TCR knockout while preserving cell viability and recovery.
  • Seamless scale-up. Performance between the low-volume Scan to high-volume Max was roughly identical (within 1–2%) without re-optimization.

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