OR06

Editing of HSPCs is an attractive strategy to treat genetic disorders and can be achieved using CRISPR/Cas9. The Cas9 induced double-stranded break (DSB) can be repaired mainly by NonHomologous End Joining (NHEJ) or Homologous Directed Repair (HDR). The latter can be hijacked to insert curative sequences delivered by an adeno-associated virus (AAV). However, HDR activity is restricted to the S/G2 cell-cycle phases, limiting targeted integration. NHEJ, instead, is always active, competing with HDR for DSB reparation. We thus tested a drug inhibiting NHEJ, aiming to favor HDR. We used Cas9-gRNA ribonucleoprotein complex, targeting the α-globin promoter, alone or with an AAV6 carrying a promoterless GFP and PGK-NGFR cassette, flanked by homology arms to the genomic target site. Different concentrations of the NHEJ inhibitor (NHEJi) were tested on HSPCs, and the efficiency of DNA insertion was evaluated. GFP and NGFR signals were analyzed by flow cytometry. NHEJi resulted in a ~2.5-fold increase in the GFP/NGFR + cells, going from 20% to 55% of the total population. This increase was confirmed by ddPCR for DNA on target integration, going from 0.2 to 1 copy per cell. Noteworthy, we reported that NHEJi could also increase the efficiency of CRISPRmediated genomic deletion, as indicated by a ddPCR measuring deletion efficiency between the two cutting sites of our single gRNA. In addition, targeted long-read sequencing is on-going to obtain a more detailed analysis of targeted integration and deletion events. Overall, these data highlight the potential of NHEJi as a promising strategy for tuning genome editing.