Hahn Florian, Mantegazza Otho, Greiner André, Hegemann Peter, Eisenhut Marion, Weber Andreas P M
Institute of Plant Biochemistry, Cluster of Excellence on Plant Science, Center for Synthetic Life Sciences, Heinrich Heine University Düsseldorf, Germany.
Institute of Biology, Experimental Biophysics, Humboldt-Universität zu Berlin Berlin, Germany.
Front Plant Sci. 2017 Jan 24;8:39. doi: 10.3389/fpls.2017.00039. eCollection 2017.
The CRISPR/Cas9 system enables precision editing of the genome of the model plant and likely of any other organism. Tools and methods for further developing and optimizing this widespread and versatile system in would hence be welcomed. Here, we designed a generic vector system that can be used to clone any sgRNA sequence in a plant T-DNA vector containing an ubiquitously expressed gene. With this vector, we explored two alternative marker systems for tracking Cas9-mediated gene-editing : () and (). confers resistance to glufosinate and is widely used as a positive selection marker; is required for the formation of trichomes. Reversion of a frameshift null allele to a functional one by Cas9-mediated gene editing yielded a higher than expected number of plants that are resistant to glufosinate. Surprisingly, many of those plants did not display reversion of the gene through the germline. We hypothesize that few revertant cells in a highly chimeric plant likely provide system-wide resistance to glufosinate and thus we suggest that BAR is not suitable as marker for tracking Cas9-mediated gene-editing. Targeting the gene for disruption with Cas9 provided clearly visible phenotypes of partially and completely glabrous plants. 50% of the analyzed T1 plants produced descendants with a chimeric phenotype and we could recover fully homozygous plants in the T3 generation with high efficiency. We propose that targeting of is suitable for assessing and optimizing Cas9-mediated gene-editing in .
CRISPR/Cas9系统能够对模式植物的基因组进行精确编辑，可能也适用于任何其他生物体的基因组编辑。因此，欢迎开发和优化这一广泛应用且功能多样的系统的工具和方法。在此，我们设计了一种通用载体系统，可用于在含有普遍表达基因的植物T-DNA载体中克隆任何sgRNA序列。利用该载体，我们探索了两种用于追踪Cas9介导的基因编辑的替代标记系统：（）和（）。赋予对草铵膦的抗性，被广泛用作阳性选择标记；是形成毛状体所必需的。通过Cas9介导的基因编辑将移码无效等位基因恢复为功能性等位基因，产生了比预期数量更多的对草铵膦具有抗性的植株。令人惊讶的是，这些植株中的许多并未通过种系显示基因的回复。我们推测，高度嵌合植物中少数回复细胞可能为全系统提供了对草铵膦的抗性，因此我们认为BAR不适宜作为追踪Cas9介导的基因编辑的标记。用Cas9靶向基因进行破坏可产生部分和完全无毛植株的明显可见表型。50%的分析T1代植株产生具有嵌合表型的后代，并且我们能够在T3代高效地获得完全纯合的植株。我们提出，靶向适用于评估和优化中的Cas9介导的基因编辑。
