Impact of CRISPR/HDR editing versus lentiviral transduction on long-term engraftment and clonal dynamics of HSPCs in rhesus macaques.

For precise genome editing via CRISPR/homology-directed repair (HDR), effective and safe editing of long-term engrafting hematopoietic stem cells (LT-HSCs) is required. The impact of HDR on true LT-HSC clonal dynamics in a relevant large animal model has not been studied. To track the output and clonality of HDR-edited cells and to provide a comparison to lentivirally transduced HSCs in vivo, we developed a competitive rhesus macaque (RM) autologous transplantation model, co-infusing HSCs transduced with a barcoded GFP-expressing lentiviral vector (LV) and HDR edited at the CD33 locus. CRISPR/HDR-edited cells showed a two-log decrease by 2 months following transplantation, with little improvement via p53 inhibition, in comparison to minimal loss of LV-transduced cells long term. HDR long-term clonality was oligoclonal in contrast to highly polyclonal LV-transduced HSCs. These results suggest marked clinically relevant differences in the impact of current genetic modification approaches on HSCs.