Symeonidi Efthymia, Regalado Julian, Schwab Rebecca, Weigel Detlef
Department of Molecular Biology, Max Planck Institute for Developmental Biology, Tübingen, Germany.
Quant Plant Biol. 2021 Jan 5;2:e1. doi: 10.1017/qpb.2020.6. eCollection 2021.
Genome editing with the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR associated protein) system allows mutagenesis of a targeted region of the genome using a Cas endonuclease and an artificial guide RNA. Both because of variable efficiency with which such mutations arise and because the repair process produces a spectrum of mutations, one needs to ascertain the genome sequence at the targeted locus for many individuals that have been subjected to mutagenesis. We provide a complete protocol for the generation of amplicons up until the identification of the exact mutations in the targeted region. CRISPR-finder can be used to process thousands of individuals in a single sequencing run. We successfully identified an mutant line in which the production of salicylic acid was impaired compared to the wild type, as expected. These features establish CRISPR-finder as a high-throughput, cost-effective and efficient genotyping method of individuals whose genomes have been targeted using the CRISPR/Cas9 system.
使用CRISPR/Cas(成簇规律间隔短回文重复序列/CRISPR相关蛋白)系统进行基因组编辑,可利用Cas核酸内切酶和人工引导RNA对基因组的靶向区域进行诱变。由于此类突变产生的效率各不相同,且修复过程会产生一系列突变,因此需要确定许多经过诱变的个体在靶向位点的基因组序列。我们提供了一个完整的方案,用于生成扩增子,直至鉴定出靶向区域的确切突变。CRISPR-finder可在一次测序运行中处理数千个个体。正如预期的那样,我们成功鉴定出了一个突变株系,与野生型相比,该株系中水杨酸的生成受到了损害。这些特性使CRISPR-finder成为一种高通量、经济高效且有效的基因分型方法,可用于对使用CRISPR/Cas9系统进行基因组靶向的个体进行基因分型。