Efficient generation of stable, heritable gene edits in wheat using CRISPR/Cas9.
Efficient generation of stable, heritable gene edits in wheat using CRISPR/Cas9.
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DOI：
10.1186/s12870-018-1433-z
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发表时间：
2018-10-03
影响因子：
5.3
通讯作者：
Wallington EJ
中科院分区：
文献类型：
--
作者：
Howells RM;Craze M;Bowden S;Wallington EJ
CRISPR/Cas9系统的使用可能被证明是作物研究中的一个有价值的工具，提供了完全敲除复杂基因组中的基因功能或通过敲除单个等位基因来精确调整基因功能的能力。我们结合使用单基因组和三基因组靶向，比较了六倍体小麦（Triticum aestivum）和二倍体大麦（Hordeum vulgare）的基因编辑。使用稳定的农杆菌介导的转化在小麦中实现了高效基因编辑，单基因组靶向指导为 11-17%，三基因组靶向指导为 5%。小麦的基因编辑被证明主要是杂合的，编辑以孟德尔方式遗传多代，并且没有观察到脱靶效应。两个物种之间的编辑比较表明，小麦中产生了更稳定、可遗传的编辑，而大麦则表现出持续的体细胞编辑。我们的工作表明，仅通过两代，即可在 36 周内获得稳定编辑的非转基因小麦品系，并且只需付出适度的努力即可实现小麦基因组内个体同源物的定向诱变，并且无需脱靶突变或不需要冗长的杂交策略。本文的在线版本 (10.1186/s12870-018-1433-z) 包含补充材料，可供授权用户使用。
The use of CRISPR/Cas9 systems could prove to be a valuable tool in crop research, providing the ability to fully knockout gene function in complex genomes or to precisely adjust gene function by knockout of individual alleles. We compare gene editing in hexaploid wheat (Triticum aestivum) with diploid barley (Hordeum vulgare), using a combination of single genome and tri-genome targeting. High efficiency gene editing, 11–17% for single genome targeted guides and 5% for tri-genome targeted guides, was achieved in wheat using stable Agrobacterium-mediated transformation. Gene editing in wheat was shown to be predominantly heterozygous, edits were inherited in a Mendelian fashion over multiple generations and no off-target effects were observed. Comparison of editing between the two species demonstrated that more stable, heritable edits were produced in wheat, whilst barley exhibited continued and somatic editing. Our work shows the potential to obtain stable edited transgene-free wheat lines in 36 weeks through only two generations and that targeted mutagenesis of individual homeologues within the wheat genome is achievable with a modest amount of effort, and without off-target mutations or the need for lengthy crossing strategies. The online version of this article (10.1186/s12870-018-1433-z) contains supplementary material, which is available to authorized users.
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27.5
作者：
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通讯作者：
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