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Author:
Date:
2016-12-05
[Abstract] The CRISPR (clustered regularly interspaced short palindromic repeats)-associated protein 9 (Cas9) has become the most broadly used and powerful tool for genome editing. Many applications of CRISPR-Cas9 require the delivery of multiple small guide RNAs (gRNAs) into the same cell in order to achieve multiplexed gene editing or regulation. Using traditional co-transfection of single gRNA expression vectors, the likelihood of delivering several gRNAs into the same cell decreases in accordance with the number of gRNAs. Thus, we have developed a method to efficiently assemble gRNA expression cassettes (2-30 gRNAs) into one single vector using a Golden-Gate assembly method (Vad-Nielsen et al., 2016). In this protocol, we describe the detailed step-by-step instructions for assembly of ...
[摘要] CRISPR（聚簇定期间隔短回文重复序列）相关蛋白9（Cas9）已成为最广泛使用和强大的基因组编辑工具。 CRISPR-Cas9的许多应用需要将多个小导向RNA（gRNA）递送到相同的细胞中以实现多重基因编辑或调节。使用单个gRNA表达载体的传统共转染，将几个gRNA递送到相同细胞中的可能性根据gRNA的数量减少。因此，我们开发了使用Golden-Gate装配方法（Vad-Nielsen等人，2016）将gRNA表达盒（2-30gRNA）有效地装配到一个单一载体中的方法。在这个协议，我们描述详细的分步指示的多路复用gRNA表达式数组的组装。在我们的表达阵列中使用的gRNA支架是由人U6启动子驱动的来自化脓性链球菌的Cas9蛋白的gRNA 1.0系统。
关键字： CRISPR，SpCas9，金门汇编，多重gRNA阵列，同时遗传操作
[背景] ...
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