Figure 3.

Gene-editing correction by the CRISPR-Cas9 system evaluated in globin mRNAs

(A) Representative example of gene-correction treatment performed by the CRISPR-Cas9 system on Th-ErPC-2 and analyzed by RT-ddPCR assay. The treatment points shown in the figure are: C− (untreated); GE Fwd (cells treated with the CRISPR-Cas9 system with fwd donor DNA); GE Rev (cells treated with the CRISPR-Cas9 system with rev donor DNA). Top and middle: 1d dot plots obtained after analysis of edited β-globin mRNA (top) or non-corrected cultures; bottom: 2d dot plot showing edited and mutated β-globin mRNA. Colored dots reported in (A) represent the events (droplets) in function of the respective FAM or HEX fluorescence. (B) Fractional abundance (in purple) related at the representative example reported in (A) is shown. (C) The fractional abundance calculated on the average of six RT-ddPCR analyses performed on ErPC cultures isolated from different β⁰39-thalassemia patients and represented by the histogram. (D) The histogram shows the relative content β⁰39 non-treated (NT) values related to the α (blue)-, β (red)-, and γ (green)-globins obtained from the treated/NT ratio for each sample analyzed. All of the data were normalized using β-actin and GAPDH as housekeeping genes.