Bachu Ravichandra, Bergareche Iñigo, Chasin Lawrence A
Department of Biological Sciences, Columbia University, New York, New York, 10027.
Biotechnol Bioeng. 2015 Oct;112(10):2154-62. doi: 10.1002/bit.25629. Epub 2015 Jul 7.
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 ∼5 kb 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.
哺乳动物细胞被广泛用于生产治疗性重组蛋白，因为这些细胞有助于准确折叠和进行翻译后修饰，而这些对于最佳活性往往至关重要。将携带感兴趣基因的质粒靶向插入哺乳动物细胞基因组以表达所需蛋白质是生产此类生物制品的关键步骤。在此我们表明，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）可通过非同源末端连接（NHEJ）有效地将约5 kb的质粒靶向插入哺乳动物基因组。我们在HEK293细胞中实现了高达0.17%的效率，在CHO细胞中实现了0.45%的效率。这项技术有望将大的外源DNA序列快速高效地插入哺乳动物细胞的预定基因组位点。
