Does cell quiescence prevent LOH induced by CRISPR? – CRISPR-SAFE

CRISPR-Cas9 is a breakthrough that has propelled regenerative medicine into a new era. Considerable progress is being made by several ongoing clinical studies using CRISPR-Cas9 nuclease in fields such as immunotherapy, regenerative medicine, and monogenic diseases. Nevertheless, a major concern is the potential genotoxicity of DNA double-strand breaks (DSB), which arise from incorrect DNA repair. We recently reported that the adverse events are more disruptive than the sole induction of insertion and deletions (Indels) at the targeted locus (Cullot et al Nat com 2019, Boutin et al Nat com, 2021). Based on novel and robust preliminary results (Cullot et al in review), we hypothesize that the cell cycle phase during which the DSB occurs could influence the rate of loss of heterozygosity (LOH). Our ANR project will (i) reinforce preliminary results by establishing ex vivo the correlation between the cell cycle slowdown and the decrease in LOH occurrence in primary cells used for clinical trials (Hematopoietic stem/progenitor cells and T-cells) (ii) determine cell culture conditions modulating the cell cycle to prevent the risk of LOH (iii) develop a temporal control of CRISPR-Cas9 nuclease genome editing by the development of novel Cas-9 (iv) evaluate the risk of LOH in vivo in hematopoietic grafts in an animal mouse model. To answer these questions, we have invented two novel convenient readout systems to easily detect LOH by flow cytometry. Analyses will be completed by single cell RNA sequencing, improved to detect all LOH type (copy neutral and copy loss LOH). This original project is a fundamental step in finding solutions to increase the safety of CRISPR technology for the development of regenerative medicine.