Highly efficient activation of endogenous gene in grape using CRISPR/dCas9-based transcriptional activators
Highly efficient activation of endogenous gene in grape using CRISPR/dCas9-based transcriptional activators
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DOI：
10.1093/hr/uhab037
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发表时间：
2022-01
影响因子：
8.7
通讯作者：
C. Ren;Huayang Li;Yanfei Liu;Shaohua Li;Zhenchang Liang
中科院分区：
文献类型：
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作者：
C. Ren;Huayang Li;Yanfei Liu;Shaohua Li;Zhenchang Liang
目的基因的过表达和敲除（或敲低）是基因功能研究中常用的两种策略。规则间隔短回文重复序列（CRISPR）/Cas9系统介导的基因敲除已应用于大多数植物物种，包括葡萄藤。然而，基于CRISPR/dCas 9（失活的Cas9）的转录激活在水果作物中仍然没有报道，尽管在拟南芥和水稻中已经记录了一些研究。在此，我们测试了两种转录激活因子VP 64和TV对葡萄内源基因的转录激活。dCas 9-VP 64和dCas 9-TV系统对于葡萄细胞中UDP-葡萄糖类黄酮糖基转移酶（UFGT）基因的转录激活都足够有效。dCas 9-VP 64系统在UFGT活化中的有效性为约1.6至5.6倍，而dCas 9-TV系统的效率为约5.7至7.2倍。此外，在葡萄藤植物中，通过使用dCas 9-TV系统实现了冷响应转录因子基因CBF 4的高效活化。CBF 4的表达在转基因植物中增加了3.7至42.3倍。与野生型植物相比，CBF 4激活的植物在冷处理后表现出较低的电解质渗漏。我们的结果证明了dCas 9-VP 64和dCas 9-TV系统在葡萄基因激活中的有效性，这将促进转录激活在这个经济上重要的物种中的应用。
Abstract Overexpression and knockout (or knockdown) of gene of interest are two commonly used strategies for gene functional study. Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system-mediated gene knockout has been applied in most plant species, including grapevine. However, CRISPR/dCas9 (deactivated Cas9)-based transcriptional activation is still unreported in fruit crops, although a few studies have been documented in Arabidopsis and rice. Here, we tested two transcriptional activators VP64 and TV for transcriptional activation of endogenous genes in grape. Both the dCas9-VP64 and dCas9-TV systems are efficient enough for transcriptional activation of the UDP-glucose flavonoid glycosyltransferases (UFGT) gene in grape cells. The effectiveness of the dCas9-VP64 system in UFGT activation was about 1.6- to 5.6-fold, while the efficiency of the dCas9-TV system was around 5.7- to 7.2-fold. Moreover, in grapevine plants, highly efficient activation of the cold-responsive transcription factor gene CBF4 was achieved by using the dCas9-TV system. The expression of CBF4 was increased 3.7- to 42.3-fold in transgenic plants. Compared with the wild-type plants, the CBF4-activated plants exhibited lower electrolyte leakage after cold treatment. Our results demonstrate the effectiveness of the dCas9-VP64 and dCas9-TV systems in gene activation in grape, which will facilitate application of transcriptional activation in this economically important species.