title: CRISPR/Cas mutagenesis of soybean and Medicago truncatula using a new web-tool and a modified Cas9 enzyme.
abstract: The CRISPR/Cas9 system is rapidly becoming the reagent of choice for targeted mutagenesis and gene editing in crop species. There are currently intense research efforts in the crop sciences to identify efficient CRISPR/Cas9 platforms to carry out targeted mutagenesis and gene editing projects. These efforts typically result in the incremental tweaking of various platform components including the identification of crop-specific promoters and terminators for optimal expression of the Cas9 enzyme and identification of promoters for expression of the CRISPR guide RNA. In this report, we demonstrate the development of an online web tool for fast identification of CRISPR/Cas9 target loci within soybean gene models, and generic DNA sequences. The web-tool described in this work can quickly identify a high number of potential CRISPR/Cas9 target sites, including restriction enzyme sites that can facilitate the detection of new mutations. In conjunction with the web tool, a soybean codon-optimized CRISPR/Cas9 platform was designed to direct double-stranded breaks to the targeted loci in hairy root transformed cells. The modified Cas9 enzyme was shown to successfully mutate target genes in somatic cells of 2 legume species, soybean and Medicago truncatula. These new tools may help facilitate targeted mutagenesis in legume and other plant species.
authors.affiliation: a Department of Agronomy and Plant Genetics ; University of Minnesota ; Saint Paul , MN USA.
authors.forename: Jean-Michel
authors.initials: JM
authors.lastname: Michno
authors.affiliation: a Department of Agronomy and Plant Genetics ; University of Minnesota ; Saint Paul , MN USA.|b School of Agronomy; Anhui Agricultural University ; Hefei , China.
authors.forename: Xiaobo
authors.initials: X
authors.lastname: Wang
authors.affiliation: a Department of Agronomy and Plant Genetics ; University of Minnesota ; Saint Paul , MN USA.
authors.forename: Junqi
authors.initials: J
authors.lastname: Liu
authors.affiliation: a Department of Agronomy and Plant Genetics ; University of Minnesota ; Saint Paul , MN USA.|c Department of Plant Pathology ; University of Minnesota ; Saint Paul , MN USA.
authors.forename: Shaun J
authors.initials: SJ
authors.lastname: Curtin
authors.affiliation: a Department of Agronomy and Plant Genetics ; University of Minnesota ; Saint Paul , MN USA.
authors.forename: Thomas Jy
authors.initials: TJ
authors.lastname: Kono
authors.affiliation: a Department of Agronomy and Plant Genetics ; University of Minnesota ; Saint Paul , MN USA.
authors.forename: Robert M
authors.initials: RM
authors.lastname: Stupar
mesh_terms: D001665:Binding Sites; D064130:CRISPR-Associated Proteins / Q000737:chemistry* / Q000235:genetics; D064113:CRISPR-Cas Systems; D017343:Genes, Plant; D005818:Genetic Engineering / Q000379:methods; D020407:Internet; D046913:Medicago truncatula / Q000235:genetics*; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D030821:Plants, Genetically Modified; D011401:Promoter Regions, Genetic; D017422:Sequence Analysis, DNA; D012984:Software; D013025:Glycine max / Q000235:genetics*
chemical_list: D064130:CRISPR-Associated Proteins
keywords: CRISPR/Cas9; Medicago; genome engineering; hairy roots; soybean; targeted mutagenesis