CRISPR-Cas targeted plasmid integration into mammalian cells via non-homologous end joining
CRISPR-Cas targeted plasmid integration into mammalian cells via non-homologous end joining
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DOI：
10.1002/bit.25629
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发表时间：
2015-10-01
影响因子：
3.8
通讯作者：
Chasin, Lawrence A.
中科院分区：
文献类型：
--
作者：
Bachu, Ravichandra;Bergareche, Inigo;Chasin, Lawrence A.
哺乳动物细胞被广泛用于生产治疗性重组蛋白，因为这些细胞促进准确折叠和翻译后修饰，这些修饰通常是最佳活性所必需的。将携带目的基因的质粒靶向插入哺乳动物细胞的基因组中以表达所需蛋白质是生产此类生物制剂的关键步骤。在这里，我们表明，使用CRISPR-Cas9系统在基因组和供体质粒中产生的位点特异性双链断裂（DSB）可以有效地用于通过非同源末端连接（NHEJ）将约5 kb质粒靶向哺乳动物基因组。我们能够在HEK 293细胞中实现高达0.17%的效率，在CHO细胞中实现高达0.45%的效率。该技术有望将大的外源DNA序列快速有效地插入哺乳动物细胞中的预定基因组位点。Biotechnol. Bioeng. 2015;112：2154-2162. (c)2015 Wiley Periodicals，Inc.
Mammalian cells are widely used for the production of therapeutic recombinant proteins, as these cells facilitate accurate folding and post-translational modifications often essential for optimum activity. Targeted insertion of a plasmid harboring a gene of interest into the genome of mammalian cells for the expression of a desired protein is a key step in production of such biologics. Here we show that a site specific double strand break (DSB) generated both in the genome and the donor plasmid using the CRISPR-Cas9 system can be efficiently used to target approximate to 5kb plasmids into mammalian genomes via nonhomologous end joining (NHEJ). We were able to achieve efficiencies of up to 0.17% in HEK293 cells and 0.45% in CHO cells. This technique holds promise for quick and efficient insertion of a large foreign DNA sequence into a predetermined genomic site in mammalian cells. Biotechnol. Bioeng. 2015;112: 2154-2162. (c) 2015 Wiley Periodicals, Inc.