250,000 cells (IPF) in 20 µL were used per electroporation. Cells were washed once in phosphate-buffered saline (PBS), and pelleted by centrifugation at 90g, 10min, at room temperature. After PBS removal, cells were re-suspended in 15.5 µL of P3 solution from a P3 Primary Cell 4D-Nucleofector X Kit (Lonza) and mixed together with 4.5 µL of RNP complex to reach a total volume of 20 µL. Electroporation was performed in 16-well Nucleocuvette strips using
program CM-138. For a double KO generation using the 4D Nucleofector system, RNPs targeting SMAD2 exon 6 and SMAD3 exon 6 were complexed in vitro as  described above. A total of 250,000 cells were washed once in PBS, and pelleted by centrifugation at 90g, 10 min, at room temperature. After PBS removal, cells were re-suspended in 11µL of P3 solution and mixed together with 9 µL of RNPs to reach a final volume of 20 µL. Electroporation was performed in 16-well Nucleocuvette Strips using program CM-138.

Expanded CD4+ T cells (n = 1,000,000) derived from peripheral blood were re-suspended in 20 µL of P3 primary Cell4D-Nucleofector X Kit Buffer (Lonza) and were then
mixed with 15 lL of RNP complex for a total of 35 µL. Electroporation was carried out using a 4D Nucleofector system in 16-well Nucleocuvette Strips with the EH-115 program.