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.
Bibliographical noteFunding Information:
Authors thank Dr Joachim Messing (Rutgers, The State University of New Jersey) for the pWI-11 plasmid containing the WDV genome and Dr Toni Wendt (Aarhus University, Denmark) for the Ubi-GFP plasmid. We acknowledge Dr Kan Wang (Iowa State University) for providing the corn ears for the isolation of immature embryos and Jade Mathre for technical assistance. We also thank Kit Leffler for editing and preparing the figures. This work was supported in part by grants to D.F.V. from the National Science Foundation (IOS-1444511and IOS-1339209), and to C.G. from the National Natural Science Foundation of China (31420103912). Javier Gil-Humanes acknowledges the Fundación Alfonso Martin Escudero for his post-doctoral fellowship.
© 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd
- DNA replicons
- genome editing
- homologous recombination
- multiplexed gene targeting
- technical advance