Targeted deletion of floral development genes in Arabidopsis with CRISPR/Cas9 using the RNA endoribonuclease Csy4 processing system
Targeted deletion of floral development genes in Arabidopsis with CRISPR/Cas9 using the RNA endoribonuclease Csy4 processing system
复制标题
使用 RNA 核糖核酸内切酶 Csy4 处理系统，通过 CRISPR/Cas9 定向删除拟南芥中的花发育基因
DOI：
10.1038/s41438-019-0179-6
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发表时间：
2019-08-21
影响因子：
8.7
通讯作者：
Zhang, Qixiang
中科院分区：
文献类型：
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作者：
Liu, Yingzhu;Gao, Yike;Zhang, Qixiang
高等植物花的形成受复杂的基因调控网络控制。转座子标记的突变系促进和维持了拟南芥花发育的研究。在这项研究中，我们报告了一个基于RNA内切核糖核酸酶Csy 4加工的CRISPR/Cas9基因组编辑系统，以诱导拟南芥花发育相关转录因子的高效和可遗传的靶向缺失。使用AP 1、SVP和TFL 1作为靶基因，用串联排列的Csy 4-sgRNA结构实现多位点和多基因突变，以在转基因系中从由Pol II启动子驱动的单个转录物表达多重sgRNA。通过使用单个二元载体产生一个或三个基因中的第一外显子和第二外显子之间的染色体片段的靶向缺失。有趣的是，位点靶向缺失的效率与使用多重sgRNA的插入缺失突变的效率相当。RT-PCR产物的DNA测序分析表明，AP 1和TFL 1的靶向缺失可导致移码突变，并引入过早的终止密码子以破坏靶基因的开放阅读框。此外，在SVP靶向缺失后没有获得RT-PCR扩增产物。此外，与野生型植物相比，靶向缺失导致突变株系中的花发育异常。AP 1和SVP突变增加植物分枝显着，而TFL 1突变体植物表现出从不定花序到确定花序的变化。因此，我们的结果表明，CRISPR/Cas9与RNA内切核糖核酸酶Csy 4加工系统是一种有效的工具，研究花发育和改善花性状快速，简单。
The formation of flowers in higher plants is controlled by complex gene regulatory networks. The study of floral development in Arabidopsis is promoted and maintained by transposon-tagged mutant lines. In this study, we report a CRISPR/Cas9 genome-editing system based on RNA endoribonuclease Csy4 processing to induce high-efficiency and inheritable targeted deletion of transcription factors involved in floral development in Arabidopsis. Using AP1, SVP, and TFL1 as the target genes, multisite and multiple-gene mutations were achieved with a tandemly arrayed Csy4-sgRNA architecture to express multiplexed sgRNAs from a single transcript driven by the Pol II promoter in transgenic lines. Targeted deletions of chromosomal fragments between the first exon and second exon in either one or three genes were generated by using a single binary vector. Interestingly, the efficiency of site-targeted deletion was comparable to that of indel mutation with the multiplexed sgRNAs. DNA sequencing analysis of RT-PCR products showed that targeted deletions of AP1 and TFL1 could lead to frameshift mutations and introduce premature stop codons to disrupt the open-reading frames of the target genes. In addition, no RT-PCR amplified product was acquired after SVP-targeted deletion. Furthermore, the targeted deletions resulted in abnormal floral development in the mutant lines compared to that of wild-type plants. AP1 and SVP mutations increased plant branching significantly, while TFL1 mutant plants displayed a change from indeterminate to determinate inflorescences. Thus, our results demonstrate that CRISPR/Cas9 with the RNA endoribonuclease Csy4 processing system is an efficient tool to study floral development and improve floral traits rapidly and simply.