Cas9:sgRNA RNP disruption of a transcriptional repressor binding motif in the γ-globin gene promoters. (A) The extended β-globin locus, showing the target BCL11A binding motif in the promoters of the genes encoding γ-globin (HBG2 and HBG1). The 13-nt HPFH deletion is indicated by a dashed black line. sgRNA-1 target is represented by a black line, with a vertical arrow indicating the predicted site of Cas9 dsDNA cleavage and the protospacer adjacent motif (PAM) sequence indicated in gray. (B) Optimization of the Cas9:sgRNA-1 ratio on indel formation, using the Neon Transfection System. RNPs were generated by incubating the indicated amounts of Cas9 and sgRNA in 5 μL of 10 mM HEPES, 150 mM NaCl for 30 min at room temperature. Here and in subsequent experiments, sgRNA-1 was chemically modified to enhance stability. The RNPs were mixed with 2 × 10⁵ CD34⁺ cells in T buffer in a final volume of 10 μL, electroporated using a Neon Transfection System at 1600 V, with 3 pulses of 10 ms, cultured for 4 days, and then analyzed for indel formation by high-throughput sequencing of PCR products generated using primers located ∼150 bp on either side of the RNP cleavage site. The graphs show the mean ± standard deviation (SD) indel frequency on the y-axis (n = 3 biological replicates). Dark blue indicates the 13-nt human HPFH mutation; light blue represents all other indels. (C) Sequence alignment of the most common mutant alleles. The sgRNA-1 sequence is underlined and bolded, the PAM sequence is in red, and the BCL11A-binding motif is highlighted in blue. Deletions are represented by dashes. The percentage of each mutation observed is shown on the right, with the NGS read counts in parentheses. LCR, locus control region.