title: High-efficiency gene targeting in hexaploid wheat using DNA replicons and CRISPR/Cas9.
abstract: The ability to edit plant genomes through gene targeting (GT) requires efficient methods to deliver both sequence-specific nucleases (SSNs) and repair templates to plant cells. This is typically achieved using Agrobacterium T-DNA, biolistics or by stably integrating nuclease-encoding cassettes and repair templates into the plant genome. In dicotyledonous plants, such as Nicotinana tabacum (tobacco) and Solanum lycopersicum (tomato), greater than 10-fold enhancements in GT frequencies have been achieved using DNA virus-based replicons. These replicons transiently amplify to high copy numbers in plant cells to deliver abundant SSNs and repair templates to achieve targeted gene modification. In the present work, we developed a replicon-based system for genome engineering of cereal crops using a deconstructed version of the wheat dwarf virus (WDV). In wheat cells, the replicons achieve a 110-fold increase in expression of a reporter gene relative to non-replicating controls. Furthermore, replicons carrying CRISPR/Cas9 nucleases and repair templates achieved GT at an endogenous ubiquitin locus at frequencies 12-fold greater than non-viral delivery methods. The use of a strong promoter to express Cas9 was critical to attain these high GT frequencies. We also demonstrate gene-targeted integration by homologous recombination (HR) in all three of the homoeoalleles (A, B and D) of the hexaploid wheat genome, and we show that with the WDV replicons, multiplexed GT within the same wheat cell can be achieved at frequencies of ~1%. In conclusion, high frequencies of GT using WDV-based DNA replicons will make it possible to edit complex cereal genomes without the need to integrate GT reagents into the genome.
authors.affiliation: Department of Genetics, Cell Biology, and Development, Center for Genome Engineering, University of Minnesota, Minneapolis, MN, 55455, USA.|Calyxt Inc., New Brighton, MN, 55112, USA.
authors.forename: Javier
authors.initials: J
authors.lastname: Gil-Humanes
authors.affiliation: State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, 100101, China.
authors.forename: Yanpeng
authors.initials: Y
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, 100101, China.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Liang
authors.affiliation: State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, 100101, China.
authors.forename: Qiwei
authors.initials: Q
authors.lastname: Shan
authors.affiliation: Institute for Sustainable Agriculture, CSIC, E-14080, Córdoba, Spain.
authors.forename: Carmen V
authors.initials: CV
authors.lastname: Ozuna
authors.affiliation: Institute for Sustainable Agriculture, CSIC, E-14080, Córdoba, Spain.
authors.forename: Susana
authors.initials: S
authors.lastname: Sánchez-León
authors.affiliation: Department of Genetics, Cell Biology, and Development, Center for Genome Engineering, University of Minnesota, Minneapolis, MN, 55455, USA.|Calyxt Inc., New Brighton, MN, 55112, USA.
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Baltes
authors.affiliation: Department of Genetics, Cell Biology, and Development, Center for Genome Engineering, University of Minnesota, Minneapolis, MN, 55455, USA.
authors.forename: Colby
authors.initials: C
authors.lastname: Starker
authors.affiliation: Institute for Sustainable Agriculture, CSIC, E-14080, Córdoba, Spain.
authors.forename: Francisco
authors.initials: F
authors.lastname: Barro
authors.affiliation: State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, 100101, China.
authors.forename: Caixia
authors.initials: C
authors.lastname: Gao
authors.affiliation: Department of Genetics, Cell Biology, and Development, Center for Genome Engineering, University of Minnesota, Minneapolis, MN, 55455, USA.
authors.forename: Daniel F
authors.initials: DF
authors.lastname: Voytas
mesh_terms: D060054:Agrobacterium / Q000235:genetics; D064113:CRISPR-Cas Systems / Q000235:genetics / Q000502:physiology*; D004269:DNA, Bacterial / Q000235:genetics; D000072669:Gene Editing; D018390:Gene Targeting / Q000379:methods*; D018551:Solanum lycopersicum / Q000235:genetics / Q000378:metabolism; D012093:Replicon / Q000235:genetics*; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism; D014908:Triticum / Q000235:genetics* / Q000378:metabolism*
chemical_list: D004269:DNA, Bacterial; C036483:T-DNA
keywords: CRISPR/Cas9; DNA replicons; Wheat; genome editing; homologous recombination; multiplexed gene targeting; technical advance