Revolutionizing plant biology: multiple ways of genome engineering by CRISPR/Cas

Simon Schiml, Holger Puchta

The precise manipulation of plant genomes relies on the induction of DNA double-strand breaks by site-specific nucleases to initiate DNA repair reactions that are either based on non-homologous end joining (NHEJ) or homologous recombination (HR). Recently, the CRISPR/Cas system emerged as the most important tool for genome engineering due to its simple structure and its applicability to a wide range of organisms. Here, we review the current status of its various applications in plants, where it is used for the successful generation of stable mutations in a steadily growing number of species through NHEJ. Furthermore, tremendous progress in plant genome engineering by HR was obtained by the setup of replicon mediated and in planta gene targeting techniques. Finally, other complex approaches that rely on the induction of more than one DNA lesion at a time such as paired nickases to avoid off-site effects or controlled genomic deletions are beginning to be applied routinely.