使用 CRISPR/Cas9 系统在哺乳动物细胞系中生成单细胞基因敲除克隆的指南。

IF 3.7 3区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY
Taeyeon Hong , Seung-Min Bae , Gwonhwa Song , Whasun Lim
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引用次数: 0

摘要

基因组编辑技术在各个研究领域发展迅速,可对细胞、植物、病毒和动物等多种生物体进行有针对性的基因组修饰。CRISPR/Cas9 系统是基因编辑的有力工具,可高精度地生成细胞和动物模型。CRISPR/Cas9 的临床潜力已被广泛报道,可应用于遗传疾病矫正、病毒复制抑制以及各种癌症的个性化或靶向治疗。在本研究中,我们提供了关于单导RNA(sgRNA)设计、将sgRNA克隆到质粒载体、通过转染进行单细胞分离以及利用新一代测序鉴定基因敲除克隆的指南。此外,我们还提供了通过新一代测序(NGS)将基因插入哺乳动物细胞系的结果,为从事人类和动物细胞系研究的人员提供了有用的信息。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Guide for generating single-cell–derived knockout clones in mammalian cell lines using the CRISPR/Cas9 system

Genome editing has developed rapidly in various research fields for targeted genome modifications in many organisms, including cells, plants, viruses, and animals. The clustered regularly interspaced short palindromic repeats-associated protein 9 system stands as a potent tool in gene editing for generating cells and animal models with high precision. The clinical potential of clustered regularly interspaced short palindromic repeats-associated protein 9 has been extensively reported, with applications in genetic disease correction, inhibition of viral replication, and personalized or targeted therapeutics for various cancers. In this study, we provide a guide on single-guide RNA design, cloning single-guide RNA into plasmid vectors, single-cell isolation via transfection, and identification of knockout clones using next-generation sequencing. In addition, by providing the results of insertion into mammalian cell lines through next-generation sequencing, we offer useful information to those conducting research on human and animal cell lines.

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来源期刊
Molecules and Cells
Molecules and Cells 生物-生化与分子生物学
CiteScore
6.60
自引率
10.50%
发文量
83
审稿时长
2.3 months
期刊介绍: Molecules and Cells is an international on-line open-access journal devoted to the advancement and dissemination of fundamental knowledge in molecular and cellular biology. It was launched in 1990 and ISO abbreviation is ''Mol. Cells''. Reports on a broad range of topics of general interest to molecular and cell biologists are published. It is published on the last day of each month by the Korean Society for Molecular and Cellular Biology.
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