Efficient and transgene-free gene targeting using Agrobacterium-mediated delivery of the CRISPR/Cas9 system in tomato
Precise genome editing technologies are rapidly emerging as prime tools for improving crop characteristics. The CRISPR/Cas9 system has proved highly versatile and efficient for inducing targeted DNA double-strand breaks (DSBs) in genomes. DNA DSBs in eukaryotes can be repaired using two different pathways: non-homologous end joining (NHEJ) or homologous recombination (HR). Repair of a DSB through NHEJ can be error prone and result in partially predicted deletions or insertions in the target sequence that can lead to gene knock-out (KO). In contrast, homology-directed repair (HDR) of a DSB through HR can be predicted and requires a DNA donor template with homologous flanking sequences that allows gene editing or knock-in (KI). The NHEJ-repair pathway is predominant in plants and has been widely used to obtain targeted KO of genes of interest. Although HDR gene editing (or KI) holds promise for crop breeding, this approach remains more difficult to master. It has a low success rate (few...
This work was supported by the French National Research Agency, (ref no. ANR11-BTBR-0001-GENIUS). The authors thank Peter Rogowsky for his efficient management of the GENIUS project and Holger Puchta and the KIT Botanisches Institut for the pDe–CAS9 plasmid. The authors thank Luc Jourdon (GAFL Unit) for his technical support for in vitro culture. The IJPB benefits from the support of the LabEx Saclay Plant Sciences-SPS (ANR-10-LABX-0040-SPS).
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