Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection
Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection
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DOI：
10.1016/j.jbiotec.2015.04.024
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发表时间：
2015-08-20
影响因子：
4.1
通讯作者：
Chesnut, Jonathan D.
中科院分区：
文献类型：
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作者：
Liang, Xiquan;Potter, Jason;Chesnut, Jonathan D.
CRISPR-Cas9 系统提供了一个高效基因组编辑平台，可实现哺乳动物细胞工程的创新应用。然而，Cas9 的递送和引导 RNA (gRNA) 的合成仍然是限制整体效率和易用性的步骤。在这里，我们描述了快速合成 gRNA 以及通过脂质体介导的转染或电穿孔将 Cas9 蛋白/gRNA 核糖核蛋白复合物 (Cas9 RNP) 递送到各种哺乳动物细胞中的方法。使用这些方法，我们报告针对单个靶点，Jurkat T 细胞中核酸酶介导的插入缺失率高达 94%，诱导多能干细胞 (iPSC) 的插入缺失率高达 87%。当我们使用这种方法在 Jurkat 细胞中进行多基因靶向时，我们发现在所得分离细胞系中分别约 93% 和 65% 实现了两基因座和三基因座插入缺失。此外，我们发现与质粒 DNA 转染相比，使用 Cas9 蛋白可以降低脱靶切割率。总之，我们提出了一个简化的细胞工程工作流程，使 gRNA 设计能够在短短四天内分析编辑的细胞，并在难以转染的细胞中实现高效的基因组调制。试剂的制备和向细胞的递送适合于高通量、多重基因组范围的细胞工程。 (C) 2015 年作者。由 Elsevier B.V. 出版
CRISPR-Cas9 systems provide a platform for high efficiency genome editing that are enabling innovative applications of mammalian cell engineering. However, the delivery of Cas9 and synthesis of guide RNA (gRNA) remain as steps that can limit overall efficiency and ease of use. Here we describe methods for rapid synthesis of gRNA and for delivery of Cas9 protein/gRNA ribonucleoprotein complexes (Cas9 RNPs) into a variety of mammalian cells through liposome-mediated transfection or electroporation. Using these methods, we report nuclease-mediated indel rates of up to 94% in Jurkat T cells and 87% in induced pluripotent stem cells (iPSC) for a single target. When we used this approach for multigene targeting in Jurkat cells we found that two-locus and three-locus indels were achieved in approximately 93% and 65% of the resulting isolated cell lines, respectively. Further, we found that the off-target cleavage rate is reduced using Cas9 protein when compared to plasmid DNA transfection. Taken together, we present a streamlined cell engineering workflow that enables gRNA design to analysis of edited cells in as little as four days and results in highly efficient genome modulation in hard-to-transfect cells. The reagent preparation and delivery to cells is amenable to high throughput, multiplexed genome-wide cell engineering. (C) 2015 The Authors. Published by Elsevier B.V.