Gene editing in K562 cells and primary CD34⁺ cells. (A) Schematic of sequence-specific gRNA#1WT and gRNA#1WR and their target genomic MPL sequence representing the protospacer-adjacent motif (PAM), double-strand break site (yellow arrowheads), and the W272R single point mutation site (T814C). DNA codons are underlined, and the repair template (RT) used to convert the W272R mutation to the WT sequence (W272R>WT) is also represented. (B) Example of in vitro digestion assay with gRNA#1WT or gRNA#1WR in the presence of their match or mismatch target sequences. Quantification of cutting efficiency was performed by using densitometry analysis. (C) Left panel shows quantification of in vitro cutting capabilities of gRNA#1WT and gRNA#1WR. Right panel shows quantification of the percentage of indel formation obtained with gRNA#1WT and gRNA#1WR when delivered as plasmid DNA or RNP complexes in K562 or CB CD34⁺ cells. (D) Control, unedited, and edited CD34⁺ cells isolated from patient II.4 were sequenced at day 5 after editing. G117T represents the K39N mutation and T814C represents the W272R mutation. Dotted magenta rectangles highlight the presence of additional overlapping sequences in edited cells for the T814C locus, indicating an off-target effect. (E) Flow cytometry analysis of anti-Mpl (CD110)-AlexaFluor-647 binding on control CD34⁺ cells, unedited patient II.4 CD34⁺ cells, or edited II.4 CD34⁺ cells at day 5 after editing. (F) In vitro megakaryocytic colony formation assay conducted in the presence of Tpo with the same cell samples used in panel E. *P < .05; **P < .005; ***P < .0001. CFU, colony-forming unit; DSB, double-strand break.