Transfection with fluorescent oligonucleotides and CRISPR-Cas9 ribonucleoproteins

For transfection optimization, about 7x105 primary memory T cells were transfected with NEON nucleofector (Invitrogen) at different voltages, pulses and widths, using the provided buffer T. A fluorescent siGLO oligonucleotide was used at the concentration of 1–2 μM. For the transfection of CRISPR-Cas9 ribonucleoproteins (RNPs), the selected oligonucleotides (Dharmacon, IDT) were mixed with the fluorescently labelled tracrRNA at a final concentration of 80 μM in 10 μl of nuclease-free duplex buffer (Dharmacon, IDT), followed by annealing by boiling and cool-down, to form the functional gRNA recognized by Streptococcus pyogenes Cas9. The annealed gRNA can be aliquoted and stored at -80°C for subsequent use. The RNP complex was instead prepared freshly immediately before transfection by mixing 7.5 μg of recombinant TrueCut Cas9 Protein v.2 (ThermoFisher) with 1.5 μl of the annealed gRNA in a total volume of 3 μl followed by incubation for 20 min at ~25°C. To improve transfection efficiency, Alt-R electroporation enhancer (Dharmacon, IDT) was added to the mix at a final concentration of 1.7 μM. About 7x105 freshly sorted CD4+ T cells were resuspended in Neon electroporation buffer T and electroporated using the 10 μl Neon transfection system kit, with one pulse at 2’200 V, width 20 ms, unless otherwise specified. Transfected cells were incubated 24 h in pre-warmed RPMI-1640 medium supplemented with 5% human serum, 1% non-essential amino acids, 1%, sodium pyruvate, 1% glutamine and 50 μM β-mercaptoethanol (complete medium without antibiotics). Transfection efficiency was determined by measuring the intracellular fluorescence of the ATTO-550-labelled tracrRNA by flow-cytometry 24 h post-transfection, compared to control, mock transfected cells (no RNP). To assess the efficiency of gene targeting at the level of protein or gDNA, T cells were cultured for at least 72 h after transfection. The sequences of the RNA oligonucleotides used for targeting are indicated in Table 1.

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