Thibault, C.; Rosier, J.; Riandiere, M.; Marin, V.; Fayet, S.; Berges, C.; Espasa, R.; Lamrissi-Garcia, I.; Trian, T.; Bui, S.; Dabernat, S.; Boutin, J.; Moreau-Gaudry, F.; Bedel, A.

Genome editing technologies create the potential for genetic studies and innovative gene therapies. Here we present new CRISPR-Cas9 tools, named ZIP editors (ZE), loaded with a single-stranded oligodeoxynucleotide (ssODN) template on the Cas ribonucleoprotein complex. The ssODN template is annealed to an extended guide RNA (gRNA) allowing its nuclear delivery at the right place, i.e. the targeted DNA cut, and at the right time. This new template import system is easy-to-design, easy-to-use, inexpensive and very versatile. It increases homology-directed repair (HDR) editing efficiency using Cas9 nuclease as demonstrated at many loci in many cell types (up to 12-fold higher HDR editing rate). Based on a heteroduplex gRNA-ssODN, it can also be used with the Cas9 nickase, resulting in HDR editing with minimal InDels, and preventing double-strand break (DSB)-mediated genotoxicity. ZE is a smart easy-to-use non-viral platform adaptable to targeted DSB (high HDR editing efficiency) or nick (high safety) to precisely model and correct a wide range of edits. It is suitable to many biological applications and could be considered for HDR-based gene therapies.