INV03

Challenges for CRISPR-Cas9 genotoxicity: detection and prevention

A BEDEL(1) S Fayet(1) C Thibault(1) M Riandiere(1) V Marin(1) I Lamrissi-Garcia(1) N Droin(2) P Brunet de la Grange(3) S Dabernat(1) J Boutin(1) F Moreau-Gaudry(1)

1:Université de Bordeaux INSERM U1312 eq BioGO; 2:Institut Gustave Roussy; 3:EFS Nouvelle Aquitaine

CRISPR-Cas9 is a breakthrough that has propelled regenerative medicine into a new era. Important advances are illustrated 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 or ineffective DNA repair and DNA damage response. The risk of genomic instability seems to be the Achilles heel of CRISPR–Cas9 nuclease. It is mandatory to find solutions to prevent these side effects.

Our team was pioneer to describe the presence of chromosomal rearrangements induced by CRISPR-Cas9 nuclease. We are developing innovative methods for detecting these unwanted events, making it possible to decipher the mechanisms involved in their appearance and to test the effectiveness of new editing protocols (using new DSB-less CRISPR tools or modifications of culture protocols). For example, we recently highlighted the role of cell cycle. G0/G1 cell cycle phase blockade during DSB drastically reduces chromosomal rearrangement rate, without impairing DSB efficacy.  Importantly, palbociclib exposure during editing prevents genotoxicity in cell line and in clinical-relevant hematopoietic stem cells. These results contribute to the understanding of genotoxicity mechanisms and the development of safe editing protocols with this powerful CRISPR-Cas9 tool for tomorrow's medicine.