Induction of Targeted Deletions in Transgenic Bread Wheat (Triticum aestivum L.) Using Customized Meganuclease

Journal Article / 2018

Youssef, D.; Nihou, A.; Partier, A.; Tassy, C.; Paul, W.; Rogowsky, P. M.; Beckert, M.; Barret, P.

Biotechnologies offer breeders good opportunities for breakthrough genetic improvements of bread wheat, one of mankind’s main food crops. Since the production ofthe first transgenic wheat, one ofthe major concerns has been the removal ofselective markers, first because ofsocietal concerns about the antibiotic resistance ofsome ofthese genes, and second because removal ofa selective marker was the first step toward retransformation using the same selection system. Site-directed nucleases are enzymes that cut genomic DNA in vivo at predefined sites. Among them, meganucleases cut DNA at predefined, long DNA (up to 24 nt) sites, thereby enabling single cuts on large genomes including the bread wheat genome (17 Gbp). In this paper, we describe for the first time the use ofa customized meganuclease to cut wheat DNA in vivo. We show that double cuts provoked the deletion of previously inserted DNA cassettes containing the DsRed reporter gene, and that in many cases, the meganuclease target site was correctly reconstituted, offering opportunities for subsequent insertion of stacked transgenes to replace the gene of selection. Moreover, perfect deletions were observed not only in the callus after transient expression ofthe meganucleases, but also in T0 transgenic wheat after stable retransformation with the meganuclease. Future prospects for the removal ofselective markers and transgene stacking are discussed.