Jin Yu, Marquardt Sebastian
Copenhagen Plant Science Centre, Department of Plant and Environmental Sciences, University of Copenhagen, Bülowsvej 21, 1870 Frederiksberg C, Denmark.
Bio Protoc. 2020 Oct 20;10(20):e3796. doi: 10.21769/BioProtoc.3796.
system directed by a gene-specific single guide RNA (sgRNA) is an effective tool for genome editing such as deletions of few bases in coding genes. However, targeted deletion of larger regions generate loss-of-function alleles that offer a straightforward starting point for functional dissections of genomic loci. We present an easy-to-use strategy including a fast cloning dual-sgRNA vector linked to efficient isolation of heritable genomic deletions to rapidly and cost-effectively generate a targeted heritable genome deletion. This step-by-step protocol includes gRNA design, cloning strategy and mutation detection for and may be adapted for other plant species.
由基因特异性单向导RNA（sgRNA）引导的系统是一种有效的基因组编辑工具，可用于对编码基因中的少数碱基进行缺失等操作。然而，对较大区域进行靶向缺失会产生功能丧失等位基因，这为基因组位点的功能剖析提供了一个直接的起点。我们提出了一种易于使用的策略，包括一个快速克隆的双sgRNA载体，并与高效分离可遗传的基因组缺失相结合，以快速且经济高效地产生靶向可遗传基因组缺失。这个分步方案包括gRNA设计、克隆策略和突变检测，并且可能适用于其他植物物种。
