Figure 2.

Genomic analysis of the CRISPR-Cas9 system in ErPC cultures

(A–C) The data relating to a representative example of ddPCR analysis of a β⁰39-thalassemia patient-derived ErPC (Th-ErPC-2) (A and B), whereas (C) shows the ddPCR average result of all ErPCs analyzed. (A) Representative results obtained after gene-correction treatment performed by the CRISPR-Cas9 system on a culture of ErPCs isolated from a β⁰39-thalassemia patient and analyzed by ddPCR assay. C−, untreated cells; GE forward (Fwd), cells treated with the CRISPR-Cas9 system with fwd donor DNA; GE reverse (Rev), cells treated with the CRISPR-Cas9 system with rev donor DNA. Top and middle: 1d dot plot showing edited and mutated β-globin sequence. Colored dots reported in (A) represent the events (droplets) in function of the respective FAM or HEX fluorescence. (B) Fractional abundance related at the representative ddPCR example reported in (A). (C) Average fractional abundance calculated from the average concentrations of six ddPCR analyses performed on ErPC cultures isolated from different β⁰39-thalassemia patients. (D) Electropherograms related to a small portion of the β-globin gene in which the β⁰39 mutation is highlighted with the mutated nucleotide T (red) and the corrected nucleotide C (blue) and obtained by DNA sequencing from the edited ErPC cultures.