Table 1.
Electroporation checklist for large gene knock-in in human T cells.
| sgRNA | 3 µL [60 µM] |
|---|---|
| Cas9 | 1 µL [40 µM] |
| sgRNA:Cas9 (molar ratio) | 4.5:1 |
| RNP incubation | 10 min, RT |
| RNP volume | 4 µL |
| Cells | 1 × 106/17 µL |
| Electroporation solution | P3 + S1 supplement (Lonza) |
| Template | 2 µg dsDNA in 3 µL |
| Homology arm length | 200–400 bp |
| Vol for electroporation | 23 µL |
| Format/program Neon | Strip/EH-115 |
| Incubation after electroporation | 30 min at 37 °C (in 80 µL of RPMI+20%FBS+IL7/15) |
| Transfer | 48-well plate (650 µL of RPMI+20%FBS+IL7/15) |
| Reactions per well after electroporation | 2 |