High‐efficiency gene targeting in hexaploid wheat using DNA replicons and CRISPR /Cas9

作者：Javier Gil‐Humanes, Yanpeng Wang, Zhen Liang, Qiwei Shan, Carmen Victoria Ozuna, Susana Sánchez‐León, Nicholas J. Baltes, Colby G. Starker, Francisco Barro, Caixia Gao, Daniel F. Voytas · 发表于：The Plant Journal · 年份：2016 · DOI：10.1111/tpj.13446 · 被引用次数：360 · 研究领域：CRISPR and Genetic Engineering、Plant Virus Research Studies、Chromosomal and Genetic Variations

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 ...