Fig 2. Gene editing of the PKLR locus in human hematopoietic progenitors.
(A) Analysis of homologous directed repair (HDR) in CFUs. CFUs from PuroR cells were picked individually. DNA from single CFUs was purified and the HDR was analyzed by nested PCR. This nested PCR was performed using the primers described in Fig 1A. Two sequential PCRs were performed with KI F2 and KI R2 primers and KI F3 and KI R3. Expected 1982bp band was evidenced in an agarose gel. ʎ DNA marker was used to determine the PCR band size. (B) Sanger sequencing of nested PCR products from two different CFUs (bottom sequences) and the theoretical sequence after knock-in (top sequence). Sequences of endogenous second intron, left homology arm and codon optimized RPK cDNA sequences were detected in the Sanger sequence, confirming the specific integration of the recombination matrix.