Fig 1. Gene editing protocol to introduce coRPK cassette in human hematopoietic progenitors.

(A) Scheme of knock-in of donor matrix in PKLR locus and the nested PCR analysis applied. (B) Protocol of gene editing of PKLR locus in hematopoietic progenitors. Cord Blood Hematopoietic progenitors (CB-CD34) where thawed and pre-stimulated for 24 hours. 1x10⁶ CB-CD34⁺ cells were nucleofected with the homologous recombination matrix (M) and PKLR specific TALEN^® (T) targeting a specific sequence in the second intron of PKLR. Then, the CB-CD34⁺ cells were expanded for 6 days and selected with 1μg/mL of puromycin for 4 additional days. (C) Colony forming ability of control (CTL) human CB-CD34⁺ cells, CB-CD34⁺ cells nucleofected with the recombination matrix (M) or with PKLR TALEN^® plus recombination matrix (TM) after puromycin selection period. (D) Analysis of CFUs after puromycin selection of TM-nucleofected CB-CD34⁺ cells. (E) Images of two representative CFUs derived from TM-nucleofected CB-CD34⁺ cells, myeloid CFU (top) and erythroid CFU (bottom). Kruskal-Wallis test was performed; P value is indicated in the figure. (n = 3; mean±SD).