title: An Agrobacterium-delivered CRISPR/Cas9 system for high-frequency targeted mutagenesis in maize.
abstract: CRISPR/Cas9 is a powerful genome editing tool in many organisms, including a number of monocots and dicots. Although the design and application of CRISPR/Cas9 is simpler compared to other nuclease-based genome editing tools, optimization requires the consideration of the DNA delivery and tissue regeneration methods for a particular species to achieve accuracy and efficiency. Here, we describe a public sector system, ISU Maize CRISPR, utilizing Agrobacterium-delivered CRISPR/Cas9 for high-frequency targeted mutagenesis in maize. This system consists of an Escherichia coli cloning vector and an Agrobacterium binary vector. It can be used to clone up to four guide RNAs for single or multiplex gene targeting. We evaluated this system for its mutagenesis frequency and heritability using four maize genes in two duplicated pairs: Argonaute 18 (ZmAgo18a and ZmAgo18b) and dihydroflavonol 4-reductase or anthocyaninless genes (a1 and a4). T0transgenic events carrying mono- or diallelic mutations of one locus and various combinations of allelic mutations of two loci occurred at rates over 70% mutants per transgenic events in both Hi-II and B104 genotypes. Through genetic segregation, null segregants carrying only the desired mutant alleles without the CRISPR transgene could be generated in T1progeny. Inheritance of an active CRISPR/Cas9 transgene leads to additional target-specific mutations in subsequent generations. Duplex infection of immature embryos by mixing two individual Agrobacterium strains harbouring different Cas9/gRNA modules can be performed for improved cost efficiency. Together, the findings demonstrate that the ISU Maize CRISPR platform is an effective and robust tool to targeted mutagenesis in maize.
authors: Department of Genetics, Development and Cell Biology, Iowa State University, Ames, IA, USA.
authors: Si Nian
authors: SN
authors: Char
authors: Department of Genetics, Development and Cell Biology, Iowa State University, Ames, IA, USA.
authors: Anjanasree K
authors: AK
authors: Neelakandan
authors: Department of Agronomy, Iowa State University, Ames, IA, USA.
authors: Hartinio
authors: H
authors: Nahampun
authors: Department of Agronomy, Iowa State University, Ames, IA, USA.
authors: Bronwyn
authors: B
authors: Frame
authors: Department of Agronomy, Iowa State University, Ames, IA, USA.
authors: Marcy
authors: M
authors: Main
authors: Department of Genetics, Development and Cell Biology, Iowa State University, Ames, IA, USA.
authors: Martin H
authors: MH
authors: Spalding
authors: Department of Genetics, Development and Cell Biology, Iowa State University, Ames, IA, USA.
authors: Philip W
authors: PW
authors: Becraft
authors: Donald Danforth Plant Science Center, St. Louis, MO, USA.
authors: Blake C
authors: BC
authors: Meyers
authors: Department of Biology, Stanford University, Stanford, CA, USA.
authors: Virginia
authors: V
authors: Walbot
authors: Department of Agronomy, Iowa State University, Ames, IA, USA.
authors: Kan
authors: K
authors: Wang
authors: Department of Genetics, Development and Cell Biology, Iowa State University, Ames, IA, USA.
authors: Bing
authors: B
authors: Yang
mesh_terms: D060054:Agrobacterium / Q000235:genetics*; D000483:Alleles; D060565:Argonaute Proteins / Q000235:genetics; D001483:Base Sequence; D064130:CRISPR-Associated Proteins / Q000378:metabolism; D064113:CRISPR-Cas Systems / Q000235:genetics*; D032461:Chromosomes, Plant; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D000072669:Gene Editing; D018390:Gene Targeting; D017343:Genes, Plant; D005822:Genetic Vectors / Q000235:genetics; D018745:Genome, Plant; D040582:Inheritance Patterns; D016296:Mutagenesis; D009154:Mutation; D030821:Plants, Genetically Modified / Q000235:genetics*; D000094704:RNA, Guide, CRISPR-Cas Systems; D003313:Zea mays / Q000235:genetics*
chemical_list: D060565:Argonaute Proteins; D064130:CRISPR-Associated Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems
keywords:  Argonaute ;  anthocyaninless ; CRISPR/Cas9; gene editing; maize; targeted mutagenesis