Fast and cloning-free CRISPR/Cas9-mediated genomic editing in mammalian cells.,Traffic

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Fast and cloning-free CRISPR/Cas9-mediated genomic editing in mammalian cells.
Traffic ( IF 3.6 ) Pub Date : 2019-10-17 , DOI: 10.1111/tra.12696
Paul T Manna ₁ , Luther J Davis ₁ , Margaret S Robinson ₁

Affiliation

CHoP-In (CRISPR/Cas9-mediated Homology-independent PCR-product integration) is a fast, non-homologous end-joining based, strategy for genomic editing in mammalian cells. There is no requirement for cloning in generation of the integration donor, instead the desired integration donor is produced as a polymerase chain reaction (PCR) product, flanked by the Cas9 recognition sequences of the target locus. When co-transfected with the cognate Cas9 and guide RNA, double strand breaks are introduced at the target genomic locus and at both ends of the PCR product. This allows incorporation into the genomic locus via hon-homologous end joining. The approach is versatile, allowing N-terminal, C-terminal or internal tag integration and gives predictable genomic integrations, as demonstrated for a selection of well characterised membrane trafficking proteins. The lack of donor vectors offers advantages over existing methods in terms of both speed and hands-on time. As such this approach will be a useful addition to the genome editing toolkit of those working in mammalian cell systems.

中文翻译：

在哺乳动物细胞中进行快速且非克隆的 CRISPR/Cas9 介导的基因组编辑。

CHoP-In（CRISPR/Cas9 介导的同源无关 PCR 产物整合）是一种快速、非同源末端连接的哺乳动物细胞基因组编辑策略。整合供体的生成不需要克隆，而是以聚合酶链式反应 (PCR) 产物的形式产生所需的整合供体，其两侧是目标基因座的 Cas9 识别序列。当与同源 Cas9 和引导 RNA 共转染时，在靶基因组基因座和 PCR 产物的两端引入双链断裂。这允许通过同源末端连接掺入基因组基因座。该方法用途广泛，允许 N 端、C 端或内部标签整合，并提供可预测的基因组整合，正如精选的特征明确的膜运输蛋白所证明的那样。与现有方法相比，缺乏供体载体在速度和操作时间方面都有优势。因此，这种方法将成为哺乳动物细胞系统工作人员基因组编辑工具包的有用补充。

更新日期：2019-10-17

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