Čermák Tomáš, Curtin Shaun J
Department of Genetics, Cell Biology and Development and Center for Genome Engineering, University of Minnesota, 1500 Gortner Avenue, Minneapolis, MN, 55455, USA.
Cereal Disease Laboratory, Agricultural Research Service, United States Department of Agriculture, St Paul, MN, 55108, USA.
Methods Mol Biol. 2017;1679:187-212. doi: 10.1007/978-1-4939-7337-8_12.
Advances in cereal transformation along with the completion of the wheat genome sequence assembly have increased the demand for tools that perform targeted and specific modifications in this crop plant. This protocol demonstrates the construction of reagents using a comprehensive genome engineering kit to create single and multiple gene "knockouts," site-specific chromosome deletions and gene replacement or "knockins" including the use of geminivirus replicons (GVRs). The reagents allow for both easy construction of simple genome engineering vectors, and "mix and match" swapping of components such as the Cas9, guide RNA and donor template cassettes for gene targeting. In addition, a web-based tool greatly streamlines vector selection, primer design, and vector construction.
随着谷物转化技术的进步以及小麦基因组序列组装的完成，人们对能够在这种作物中进行靶向和特异性修饰的工具的需求日益增加。本方案展示了使用一套全面的基因组工程试剂盒构建试剂，以创建单基因和多基因“敲除”、位点特异性染色体缺失以及基因替换或“敲入”，包括使用双生病毒复制子（GVR）。这些试剂既便于构建简单的基因组工程载体，又能对用于基因靶向的组件（如Cas9、引导RNA和供体模板盒）进行“混合搭配”交换。此外，一个基于网络的工具极大地简化了载体选择、引物设计和载体构建。
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