Biallelic Gene Targeting in Rice
Biallelic Gene Targeting in Rice
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DOI：
10.1104/pp.15.01663
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发表时间：
2016-02-01
期刊：
影响因子：
7.4
通讯作者：
Toki, Seiichi
中科院分区：
文献类型：
--
作者：
Endo, Masaki;Mikami, Masafumi;Toki, Seiichi
序列特异性核酸酶（SSN）已成功地用于许多生物体中的同源定向修复（HDR）介导的基因打靶（GT）。然而，由于HDR模板和SSN向植物细胞核中的低效递送，植物中断裂诱导的GT仍然具有挑战性。在包括水稻在内的许多植物中，农杆菌介导的转化是最实用的转化手段，因为与物理转化系统如聚乙二醇、电穿孔或基因枪相比，这种生物转化系统可以递送更长和更完整的DNA有效载荷，并且片段化DNA的掺入更少。在用农杆菌感染后，在共培养期间连续发生转移DNA（T-DNA）到细胞核的转移和其整合到植物基因组中，因此对靶基因上的DNA双链断裂（DSB）的诱导进行定时以与HDR模板的递送一致是至关重要的。为了同步DSB诱导和HDR模板的递送，我们将Cas9表达构建体和携带HDR模板的GT载体与靶向水稻乙酰乳酸合酶（ALS）基因的向导RNA（gRNA）单独或顺序地转化到水稻愈伤组织中。当靶向ALS的gRNA从含有HDR模板的双链T-DNA瞬时转录时，通过组装的Cas9/gRNA复合物在ALS基因座中诱导DSB，并刺激同源重组。与我们的预期相反，Cas9表达和非表达细胞之间的GT效率没有很大差异。然而，当在GT实验之前用Cas9转化靶向DNA连接酶4的gRNA时，GT效率显著增加，并且获得了在ALS基因座处表现出双等位基因GT的多于一个品系。
Sequence-specific nucleases (SSNs) have been used successfully in homology-directed repair (HDR)-mediated gene targeting (GT) in many organisms. However, break-induced GT in plants remains challenging due to inefficient delivery of HDR templates and SSNs into plant nuclei. In many plants, including rice, Agrobacterium-mediated transformation is the most practical means of transformation because this biotic transformation system can deliver longer and more intact DNA payloads with less incorporation of fragmented DNA compared with physical transformation systems such as polyethylene glycol, electroporation, or biolistics. Following infection with Agrobacterium, transfer of transfer DNA (T-DNA) to the nucleus and its integration into the plant genome occur consecutively during cocultivation, thus timing the induction of DNA double-strand breaks (DSBs) on the target gene to coincide with the delivery of the HDR template is crucial. To synchronize DSB induction and delivery of the HDR template, we transformed a Cas9 expression construct and GT vector harboring the HDR template with guide RNAs (gRNAs) targeting the rice acetolactate synthase (ALS) gene either separately or sequentially into rice calli. When gRNAs targeting ALS were transcribed transiently from double-stranded T-DNA containing the HDR template, DSBs were induced in the ALS locus by the assembled Cas9/gRNA complex and homologous recombination was stimulated. Contrary to our expectations, there was no great difference in GT efficiency between Cas9-expressing and nonexpressing cells. However, when gRNA targeting DNA ligase 4 was transformed with Cas9 prior to the GT experiment, GT efficiency increased dramatically and more than one line exhibiting biallelic GT at the ALS locus was obtained.