Cas9-Mediated Targeted Mutagenesis in Plants.
Cas9-Mediated Targeted Mutagenesis in Plants.
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DOI：
10.1007/978-1-0716-1791-5_1
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发表时间：
2022-01-01
期刊：
影响因子：
--
通讯作者：
Patron, Nicola J
中科院分区：
文献类型：
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作者：
Dudley, Quentin M;Raitskin, Oleg;Patron, Nicola J
基因组工程技术使得能够在植物基因组中的几乎任何位置诱导靶向突变。特别是，Cas9 核酸酶使用易于重新编码的 RNA 引导来靶向用户定义的序列并产生双链断裂 (DSB)，然后由细胞的内源修复机制进行修复。不正确的修复会导致靶标发生突变。当靶标位于编码序列中时，通常会导致功能丧失突变。在本章中，我们描述了一种快速设计和组装植物的 RNA 引导 Cas9 构建体的方法，并使用农杆菌介导的瞬时表达和 PEG 介导的 DNA 递送到原生质体来测试其在快速测定中诱导预期目标突变的能力，后者可以适应多种植物物种。我们描述了一种基于 PCR 的突变检测方法，并概述了将 Cas9 转基因与目标突变分离所需的步骤，从而能够生产无转基因的突变植物。这些技术适用于一系列植物物种，并应加速 Cas-9 介导的基因组工程在基础植物科学和作物开发中的应用。
Genome engineering technologies enable targeted mutations to be induced at almost any location in plant genomes. In particular, Cas9 nucleases use easily recoded RNA guides to target user-defined sequences and generate double-stranded breaks (DSB) that are then repaired by the cell's endogenous repair mechanisms. Incorrect repair results in mutations at the target. When the targets are in coding sequences, this often results in loss-of-function mutations. In this chapter, we describe a method to rapidly design and assemble RNA-guided Cas9 constructs for plants and test their ability to induce mutations at their intended targets in rapid assays using both Agrobacterium-mediated transient expression and PEG-mediated DNA delivery to protoplasts, the latter of which can be adapted to a wide range of plant species. We describe a PCR-based method for detecting mutagenesis and outline the steps required to segregate the Cas9 transgene from the targeted mutation to enable the production of transgene-free mutated plants. These techniques are amenable to a range of plant species and should accelerate the application of Cas-9-mediated genome engineering for basic plant science as well as crop development.