A modular cloning toolkit for genome editing in plants
A modular cloning toolkit for genome editing in plants
复制标题
用于植物基因组编辑的模块化克隆工具包
DOI：
10.1101/738021
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发表时间：
2019
期刊：
影响因子：
--
通讯作者：
Hahn F
中科院分区：
文献类型：
--
作者：
Hahn F
CRISPR/Cas最近已成为包括植物在内的各种生物体中广泛使用的基因组编辑工具。应用CRISPR/Cas通常需要将多个表达单位递送到植物中，因此需要快速简便的克隆程序。基于Golden Gate（GG）方法的模块化克隆（MoClo）使得能够开发具有标准化遗传部分（例如启动子、编码序列或终止子）的克隆系统，所述标准化遗传部分可以容易地互换并组装成表达单元，结果在这里，我们提出了一个扩展的克隆工具包，其中包含103个模块，编码一个各种基于CRISPR/Cas的核酸酶及其相应的指导RNA骨架。除其他组件外，该工具包还包括许多启动子，这些启动子允许CRISPR/Cas核酸酶（或任何其他编码序列）及其向导RNA在单子叶植物和双子叶植物中表达。作为工具包的一部分，我们提出了一组模块，可以快速简便地组装tRNA-sgRNA多顺反子单元，而无需PCR步骤。我们还证明了我们的tRNA-sgRNA系统在小麦原生质体中是有功能的。ConclusionsWe相信所提出的CRISPR/Cas工具包是一个很好的资源，将有助于更广泛地采用CRISPR/Cas基因组编辑技术和模块化克隆的研究人员在整个植物科学界。
BackgroundCRISPR/Cas has recently become a widely used genome editing tool in various organisms, including plants. Applying CRISPR/Cas often requires delivering multiple expression units into plant and hence there is a need for a quick and easy cloning procedure. The modular cloning (MoClo), based on the Golden Gate (GG) method, has enabled development of cloning systems with standardised genetic parts, e.g. promoters, coding sequences or terminators, that can be easily interchanged and assembled into expression units, which in their own turn can be further assembled into higher order multigene constructs.ResultsHere we present an expanded cloning toolkit that contains 103 modules encoding a variety of CRISPR/Cas-based nucleases and their corresponding guide RNA backbones. Among other components, the toolkit includes a number of promoters that allow expression of CRISPR/Cas nucleases (or any other coding sequences) and their guide RNAs in monocots and dicots. As part of the toolkit, we present a set of modules that enable quick and facile assembly of tRNA-sgRNA polycistronic units without a PCR step involved. We also demonstrate that our tRNA-sgRNA system is functional in wheat protoplasts.ConclusionsWe believe the presented CRISPR/Cas toolkit is a great resource that will contribute towards wider adoption of the CRISPR/Cas genome editing technology and modular cloning by researchers across the plant science community.