OR02

Zip Editing: an easy-to-use tool to increase CRISPR-Cas9 HDR-editing efficiency

C Thibault(1) J Rosier(1) V Marin(1,2) M Riandière(1) S Fayet(1) J Boutin(1,2) I Lamrissi-Garcia(1) T Trian(3) F Moreau-Gaudry(1,2) A Bedel(1,2)

1:BRIC-BoRdeaux Institute of oncology, Inserm / University of Bordeaux, Bordeaux, 33000, France; 2:CHU de Bordeaux, Biochemistry Laboratory, 33000 Bordeaux, France.; 3:CRCTB-Centre de Recherche Cardio-Thoracique de Bordeaux, Inserm / University of Bordeaux, 33600, Pessac, France

Genome editing using CRISPR-Cas9 holds great promise in the field of gene therapy. However, the use of CRISPR-Cas9 for precise genome editing through HDR repair pathways remains a challenge. This is mostly due to the lack of availability of the exogenous template at the site of editing at the moment of the repair. To increase the presence of the exogenous template delivered as single-stranded oligodeoxynucleotides (ssODN), we develop ZIP-Editing (ZE), a new tool that brings the ssODN with the RNP complex by annealing an extension of the ssODN template with an extension of the gRNA (patent in progress). We tested ZE in different cell lines and primary cells (including human fibroblasts and HSPCs) to edit several loci (eGFP, CFTR, UROS and HBB) with the nuclease and nickase Cas9s to challenge its universality. Thus, we demonstrated that this system, very easy to design and that uses only commercially available components, increases HDR-editing efficiency up to 12-fold as compared to a condition where ssODN template is not annealed to the gRNA. These results position ZE as a new tool to precisely edit the genome and as part of the gene editing toolbox for treating or modelling diseases. Moreover, ZE is a safer tool because it does not rely on the modification of the cell cycle, the opening of the chromatin nor the modulation of the DNA repair pathways, which can have deleterious genotoxic effects and are a major safety concern in the development of gene therapies.