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CRISPR 查找器:一种用于识别成功编辑个体的高通量且经济高效的方法。

CRISPR-finder: A high throughput and cost-effective method to identify successfully edited individuals.

作者信息

Symeonidi Efthymia, Regalado Julian, Schwab Rebecca, Weigel Detlef

机构信息

Department of Molecular Biology, Max Planck Institute for Developmental Biology, Tübingen, Germany.

出版信息

Quant Plant Biol. 2021 Jan 5;2:e1. doi: 10.1017/qpb.2020.6. eCollection 2021.

Abstract

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.

摘要

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。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/fb3a/10095899/b50c5e76045d/S2632882820000065_fig1.jpg

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