title: From classical mutagenesis to nuclease-based breeding - directing natural DNA repair for a natural end-product.
abstract: Production of mutants of crop plants by the use of chemical or physical genotoxins has a long tradition. These factors induce the natural DNA repair machinery to repair damage in an error-prone way. In the case of radiation, multiple double-strand breaks (DSBs) are induced randomly in the genome, leading in very rare cases to a desirable phenotype. In recent years the use of synthetic, site-directed nucleases (SDNs) - also referred to as sequence-specific nucleases - like the CRISPR/Cas system has enabled scientists to use exactly the same naturally occurring DNA repair mechanisms for the controlled induction of genomic changes at pre-defined sites in plant genomes. As these changes are not necessarily associated with the permanent integration of foreign DNA, the obtained organisms per se cannot be regarded as genetically modified as there is no way to distinguish them from natural variants. This applies to changes induced by DSBs as well as single-strand breaks, and involves repair by non-homologous end-joining and homologous recombination. The recent development of SDN-based 'DNA-free' approaches makes mutagenesis strategies in classical breeding indistinguishable from SDN-derived targeted genome modifications, even in regard to current regulatory rules. With the advent of new SDN technologies, much faster and more precise genome editing becomes available at reasonable cost, and potentially without requiring time-consuming deregulation of newly created phenotypes. This review will focus on classical mutagenesis breeding and the application of newly developed SDNs in order to emphasize similarities in the context of the regulatory situation for genetically modified crop plants.
authors.affiliation: Botanical Institute, Molecular Biology and Biochemistry, Karlsruhe Institute of Technology, PO 6980, 76049, Karlsruhe, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Pacher
authors.affiliation: Botanical Institute, Molecular Biology and Biochemistry, Karlsruhe Institute of Technology, PO 6980, 76049, Karlsruhe, Germany.
authors.forename: Holger
authors.identifier: http://orcid.org/0000-0003-1073-8546
authors.initials: H
authors.lastname: Puchta
mesh_terms: D004260:DNA Repair / Q000235:genetics*; D018744:DNA, Plant / Q000235:genetics*; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism; D005818:Genetic Engineering / Q000379:methods; D018745:Genome, Plant / Q000235:genetics
chemical_list: D018744:DNA, Plant; D004720:Endonucleases
keywords: gene editing; genome engineering; non-homologous end-joining; synthetic DNA nucleases