title: CRISPR/Cas-Mediated Site-Specific Mutagenesis in Arabidopsis thaliana Using Cas9 Nucleases and Paired Nickases.
abstract: The CRISPR/Cas system has recently become the most important tool for genome engineering due to its simple architecture that allows for rapidly changing the target sequence and its applicability to organisms throughout all kingdoms of life. The need for an easy-to-use and reliable nuclease is especially high in plant research, as precise genome modifications are almost impossible to achieve by Agrobacterium-mediated transformation and the regeneration of plants from protoplast cultures is very labor intensive. Here, we describe the application of the Cas9 nuclease to Arabidopsis thaliana for the induction of heritable targeted mutations, which may also be used for other plant species. To cover the concern for off-target activity, we also describe the generation of stable mutants using paired Cas9 nickases.
authors.affiliation: Botanical Institute II, Karlsruhe Institute of Technology, POB 6980, 76049, Karlsruhe, Germany.
authors.forename: Simon
authors.initials: S
authors.lastname: Schiml
authors.affiliation: Department of Plant Biology, Carnegie Institution for Science, 260 Panama St, Stanford, CA, 94305, USA.
authors.forename: Friedrich
authors.initials: F
authors.lastname: Fauser
authors.affiliation: Botanical Institute II, Karlsruhe Institute of Technology, POB 6980, 76049, Karlsruhe, Germany. holger.puchta@kit.edu.
authors.forename: Holger
authors.initials: H
authors.lastname: Puchta
mesh_terms: D017360:Arabidopsis / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D000076987:CRISPR-Associated Protein 9; D064113:CRISPR-Cas Systems; D003001:Cloning, Molecular / Q000379:methods*; D003850:Deoxyribonuclease I / Q000235:genetics / Q000378:metabolism; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D009154:Mutation; D000094704:RNA, Guide, CRISPR-Cas Systems
chemical_list: D001426:Bacterial Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems; D000076987:CRISPR-Associated Protein 9; C000606107:Cas9 endonuclease Streptococcus pyogenes; D004720:Endonucleases; D003850:Deoxyribonuclease I
keywords: Double-strand break repair; Engineered nucleases; Gene technology; Genome engineering; Targeted mutagenesis