Precise Gene Editing Preserves Hematopoietic Stem Cell Function Following Transient p53-Mediate DNA Damage Response [Amplicon-seq]

We evaluated transcriptional changes elicited by targeting AAVS1 and IL2RG genes and we found that the impact of one or few nuclease-induced DNA DSBs is minor and mainly limited to genes belonging to the p53 pathway. The same pathway was elicited by gene editing at two distinct genomic loci and was more pronounced in primitive vs. progenitor cells. Overall design: We analysed in vitro bulk population of IL2RG-edited HSPCs in presence or not of the transient expression of the p53 inhibitor GSE56. Cells were harvested 72h after gene editing procedure and processed for genomic DNA extraction and target sequencing of a 180-bp amplicon within the integrated corrective IL2RG cDNA. Similarly, we performed target sequencing by specifically amplifying the same cDNA region from peripheral blood mononuclear cells of immunodeficient NSG mice 21 weeks after transplant of HSPC edited in presence or not of GSE56.