CRISPR-based tools for targeted transcriptional and epigenetic regulation in plants
CRISPR-based tools for targeted transcriptional and epigenetic regulation in plants
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DOI：
10.1371/journal.pone.0222778
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发表时间：
2019-09-26
期刊：
影响因子：
3.7
通讯作者：
Schmid, Markus
中科院分区：
文献类型：
--
作者：
Lee, Joanne E.;Neumann, Manuela;Schmid, Markus
可编程的基因调节因子可以反式调节选定靶标的活性，是探测和操纵基因功能的有用工具。CRISPR技术为基因靶向提供了一种方便的方法，该方法也可以适用于多重和其他修饰，以通过一系列不同的效应子实现强调控。我们生成了一个用于植物中基于CRISPR/dCas 9的靶向基因调控的载体工具箱，用先前描述的MS 2系统进行修饰以放大调控的强度，并使用基于Golden Gate的克隆来实现快速载体组装，在启动子，效应子和靶标的选择方面具有高度的灵活性。我们测试了系统使用花调节基因座T（FT）作为目标和一系列不同的效应域，包括转录激活因子VP 64，H3 K27乙酰转移酶p300和H3 K9甲基转移酶KRYPTONITE。当转化到拟南芥中时，几种构建体引起与FT表达和/或表观遗传状态的变化相关的开花时间表型改变，从而证明了该系统的有效性。MS 2-CRISPR/dCas 9系统可用于调节植物中特定靶基因的转录活性和表观遗传状态，并提供了一种通用工具，可以轻松地与不同靶标和调节类型一起用于一系列应用。
Programmable gene regulators that can modulate the activity of selected targets in trans are a useful tool for probing and manipulating gene function. CRISPR technology provides a convenient method for gene targeting that can also be adapted for multiplexing and other modifications to enable strong regulation by a range of different effectors. We generated a vector toolbox for CRISPR/dCas9-based targeted gene regulation in plants, modified with the previously described MS2 system to amplify the strength of regulation, and using Golden Gate-based cloning to enable rapid vector assembly with a high degree of flexibility in the choice of promoters, effectors and targets. We tested the system using the floral regulator FLOWERING LOCUS T (FT) as a target and a range of different effector domains including the transcriptional activator VP64, the H3K27 acetyltransferase p300 and the H3K9 methyltransferase KRYPTONITE. When transformed into Arabidopsis thaliana, several of the constructs caused altered flowering time phenotypes that were associated with changes in FT expression and/or epigenetic status, thus demonstrating the effectiveness of the system. The MS2-CRISPR/dCas9 system can be used to modulate transcriptional activity and epigenetic status of specific target genes in plants, and provides a versatile tool that can easily be used with different targets and types of regulation for a range of applications.