Electroporation of U2OS cells
RNP electroporation experiments in U2OS and HEK293 minigeneexpressing cells were performed using the Lonza 4D-Nucleofector (Lonza). RNP complexes were assembled in a final volume of 5 µL using 120 pmol previously annealed crRNA-tracrRNA (IDT) duplex and 100 pmol purified SpCas9. Complexes were mixed with
2 x10^5 U2OS cells re-suspended in 20 µL SE buffer and electroporated using the CM-104 program. Genomic DNA was extracted 48 h post-electroporation to assess editing efficiency.

For RNP and ssODN delivery in HDR experiments, 120 pmol Cas9 protein and 150 pmol assembled crRNA-tracrRNA (IDT) were incubated at room temperature for 10–20 min, mixed with 120 pmol ssODN (IDT) and 120 pmol Alt-R Ca9 Electroporation Enhancer (IDT), and electroporated in a total of 2 x 10^5 U2OS or HEK293 stably expressing mutated CFTR minigenes in SE buffer (programs CM-104 and CM-130, respectively). ssODN donor sequences are reported in Table S1.

Electroporation of CD34+ cells
sgRNA containing both crRNA and tracrRNA sequences was obtained from Synthego (Table S1). RNP complexes were assembled in a final volume of 2.3–2.8µL using 180 pmol sgRNA and 90 pmol purified SpCas9. CD34+ cells (2 x 10^5 cells/condition) were transfected in the presence of 180 pmol Alt-R Cas9 Electroporation Enhancer (IDT). We used the Lonza 4D-Nucleofector (Lonza), the P3 Primary Cell 4D-Nucleofector X Kit S (Lonza), and the CA137 program. After transfection, cells were kept in the same medium for 6 days. Genomic DNA was extracted 6 days post-electroporation, and the editing was assessed by deep-sequencing (Table S1).

OT evaluation
GUIDE-seq experiments were performed as previously described.22 Briefly, 2 x 10^5 U2OS cells were electroporated with the Lonza 4D-Nucleofector (Lonza) DN-100 program using the Cas9-gRNA RNP and adding 50 pmol dsODNs (crRNAs in Table S1); cells treated using WT SpCas9 and double-strand ODNs (dsODNs) were used
as negative control.