Genome Editing in Plants using Custom-Designed Nucleases

The precise editing of plant genomes, including both site-directed mutagenesis as well as the site-specific integration of DNA molecules containing sequence with homology to a target gene (gene targeting), have great importance for basic and applied biology. Site-specific modifications are stimulated by the mechanisms used to repair double-strand DNA breaks at the target locus. Consequently, recent advances in the development of sequence-specific, custom-designed nucleases are promising for overcoming the otherwise extremely low efficiency of genome editing in plants. We have used the A. thaliana CRU3 gene as a target for site-directed mutagenesis and gene targeting. We have designed nucleases, namely transcription activator-like effector nuclease (TALENs) and nucleases based on the CRISPR/Cas system, to cleave different sites within CRU3. We compared their cleavage activities using an in-planta assay for the quantitative analysis of custom-designed nucleases. We found a high variation in the cleavage efficiency between the different nucleases, with generally a significantly higher activity for CRISPR/Cas nucleases. Moreover, using the CRISPR/Cas system, we were able to stimulate and detect precise modifications at our endogenous CRU3 target.