For both cell types, electroporation was performed using a 4D-Nucleofector system (Lonza) and cells were transferred in a single well of a 96-wells plate (Sarstedt). For K562 editing, 500 000 cells were electroporated using the SF Cell line 4D-Nucleofector Kit (Lonza) and the FF-100 program while for Jurkat cells editing, 500 000 cells were electroporated using the SE Cell line 4D-Nucleofector Kit (Lonza) and the CK-116 program. When using a recombinant AAV to deliver the repair template, the corresponding plasmid was omitted from the electroporation mix and K562 cells were transduced immediately after electroporation using 1 × 10^4 transducing unit (TU) per cell of an AAV2/6 containing the repair template produced as previously described (16).

Gene editing in human primary cells: PBMCs were activated 4 h after thawing using ImmunoCult™ Human CD3/CD28/CD2 T Cell Activator (5 µg/l × 10^6; STEMCELL Technologies) and IL-2 (100U/l; Immunotools), and kept at concentration of 2 × 10^6 cells/ml. Three days post-activation, 1 × 10^6 PBMCs were electroporated with a 4D-Nucleofector system Lonza) using the P3 Primary Cell 4D-Nucleofector Kit (Lonza) and the EO-115 program. The DNA mixture contained 30 pmol of the nuclease expressing mRNA transcribed in vitro as previously described (24), 112.5 pmol of sgRNA (Synthego) and 25 pmol of ssODN (Integrated DNA Technologies). After electroporation, complete RPMI medium supplemented with IL-2 (1000 U/ml; Immunotools) was used to recover the cells before culturing them in a 96-well U-shaped-bottom plate (Falcon). Cells were harvested 5 days post electroporation and the genomic DNA was extracted using the NucleoSpin®Tissue gDNA extraction kit (Machery Nagel) following the manufacture’s procedure and resuspended in 40 µl of Nucleasefree water. 200 000 HSPCs were electroporated with a 4D Nucleofector system (Lonza) using the P3 Primary Cell 4D Nucleofector Kit (Lonza) and the CA-137 program with a electroporation mix containing 30 pmol of nuclease expressing mRNA transcribed in vitro as previously described (25), 112.5 pmol of sgRNA (Synthego) and 50 pmol of the respective ODN (Integrated DNA Technologies). After electroporation, cells were recovered and cultured in a 96-well plate. Cells were harvested 2 days post electroporation and their genomic DNA was extracted using the NucleoSpin ® Tissue gDNA extraction kit (Machery Nagel) following the manufacture’s procedure and resuspended in 40 µl of Nuclease-free water.