利用CRISPR-Cas在硅藻中进行基因组编辑以诱导精确的双等位基因缺失
Genome Editing in Diatoms Using CRISPR-Cas to Induce Precise Bi-allelic Deletions.
作者信息
Hopes Amanda, Nekrasov Vladimir, Belshaw Nigel, Grouneva Irina, Kamoun Sophien, Mock Thomas
机构信息
School of Environmental Sciences, University of East Anglia, Norwich Research Park, Norwich, UK.
Rothamsted Research, West Common, Harpenden, Hertfordshire, UK.
出版信息
Bio Protoc. 2017 Dec 5;7(23):e2625. doi: 10.21769/BioProtoc.2625.
Abstract
Genome editing in diatoms has recently been established for the model species and . The present protocol, although developed for , can be modified to edit any diatom genome as we utilize the flexible, modular Golden Gate cloning system. The main steps include how to design a construct using Golden Gate cloning for targeting two sites, allowing a precise deletion to be introduced into the target gene. The transformation protocol is explained, as are the methods for screening using band shift assay and/or restriction site loss.
摘要
硅藻的基因组编辑最近已在模式物种[物种名称1]和[物种名称2]中得以实现。本方案虽为[物种名称3]开发，但可进行修改以编辑任何硅藻基因组，因为我们使用的是灵活的模块化金门克隆系统。主要步骤包括如何使用金门克隆设计一个针对两个位点的构建体，以便将精确的缺失引入目标基因。文中解释了转化方案，以及使用带移测定和/或限制性位点缺失进行筛选的方法。
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