Federal Research Center Institute of Cytology and Genetics, Siberian Branch of Russian Academy of Sciences, pr. Lavrentieva 10, 630090 Novosibirsk, Russia.
Cells. 2021 Aug 19;10(8):2137. doi: 10.3390/cells10082137.
Targeted DNA integration into known locations in the genome has potential advantages over the random insertional events typically achieved using conventional means of genetic modification. We investigated the possibility of obtaining a suspension cell culture of carrying a site-specific integration of a target gene encoding modified human interferon (dIFN) using endonuclease Cas9. For the targeted insertion, we selected the region of the histone H3.3 gene () with a high constitutive level of expression. Our results indicated that Cas9-induced DNA integration occurred with the highest frequency with the construction with donor DNA surrounded by homology arms and Cas9 endonuclease recognition sites. Among the monoclones of the four cell lines with knock-in studied, there is high heterogeneity in the level of expression and accumulation of the target protein. The accumulation of dIFN protein in cell lines with targeted insertions into the target region of the gene does not statistically differ from the level of accumulation of dIFN protein in the group of lines with random integration of the transgene. However, one among the monoclonal lines with knock-in has a dIFN accumulation level above 2% of TSP, which is very high.
与使用传统遗传修饰方法通常实现的随机插入事件相比，将靶向 DNA 整合到基因组中的已知位置具有潜在优势。我们研究了使用内切酶 Cas9 获得携带靶向基因（编码修饰的人干扰素（dIFN）的定点整合的悬浮细胞培养物的可能性。对于靶向插入，我们选择了组蛋白 H3.3 基因（）的区域，该区域具有高组成型表达水平。我们的结果表明，带有供体 DNA 周围同源臂和 Cas9 内切酶识别位点的构建体与 Cas9 诱导的 DNA 整合发生的频率最高。在所研究的四个带有基因敲入的细胞系的单克隆中，目标蛋白的表达和积累水平存在高度异质性。在靶向插入到基因的靶区域的细胞系中，dIFN 蛋白的积累水平与随机整合转基因的蛋白积累水平没有统计学差异。然而，在带有基因敲入的单克隆系中，有一个系的 dIFN 积累水平超过 TSP 的 2%，这非常高。
