title: A Split Staphylococcus aureus Cas9 as a Compact Genome-Editing Tool in Plants.
abstract: Split-protein methods-where a protein is split into two inactive fragments that must re-assemble to form an active protein-can be used to regulate the activity of a given protein and reduce the size of gene transcription units. Here, we show that a Staphylococcus aureus Cas9 (SaCas9) can be split, and that split-SaCas9 expressed from Agrobacterium can induce targeted mutagenesis in Nicotiana benthamiana. Since SaCas9 is smaller than the more commonly used Cas9 derived from Streptococcus pyogenes, the split-SaCas9 provides the smallest tool yet for clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) plant genome editing. Both sets of split-SaCas9 (_430N/431C and _739N/740C) exhibited genome-editing activity, and the activity of split-SaCas9_739N/740C was almost the same as that of full-length SaCas9. This result indicates that split-SaCas9_739N/740C is suitable for use in targeted mutagenesis. We also show that the split-SaCas9 fragment expressed from Tomato mosaic virus could induce targeted mutagenesis together with another fragment expressed from Agrobacterium, suggesting that a split-SaCas9 system using a plant virus vector is a promising tool for integration-free plant genome editing. Split-SaCas9 has the potential to regulate CRISPR/Cas9-mediated genome editing activity in plant cells both temporally and spatially.
authors.affiliation: Plant Genome Engineering Research Unit, Institute of Agrobiological Sciences, National Agriculture and Food Research Organization, Kannondai, Tsukuba, Ibaraki, Japan.
authors.forename: Hidetaka
authors.initials: H
authors.lastname: Kaya
authors.affiliation: Plant and Microbial Research Unit, Institute of Agrobiological Sciences, National Agriculture and Food Research Organization, Kannondai, Tsukuba, Ibaraki, Japan.
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Ishibashi
authors.affiliation: Plant Genome Engineering Research Unit, Institute of Agrobiological Sciences, National Agriculture and Food Research Organization, Kannondai, Tsukuba, Ibaraki, Japan.|Graduate School of Nanobioscience, Yokohama City University, Seto, Yokohama, Kanagawa, Japan.|Kihara Institute for Biological Research, Yokohama City University, Seto, Yokohama, Kanagawa, Japan.
authors.forename: Seiichi
authors.initials: S
authors.lastname: Toki
mesh_terms: D060054:Agrobacterium / Q000235:genetics; D004720:Endonucleases / Q000235:genetics; D000072669:Gene Editing / Q000379:methods*; D018506:Gene Expression Regulation, Plant; D018745:Genome, Plant; D016296:Mutagenesis; D018515:Plant Leaves / Q000235:genetics; D030821:Plants, Genetically Modified; D013211:Staphylococcus aureus / Q000235:genetics*; D014026:Nicotiana / Q000235:genetics*
chemical_list: D004720:Endonucleases
keywords: CRISPR/Cas9; Split-SaCas9; Staphylococcus aureus; Tomato mosaic virus