Karnan Sivasundaram, Ota Akinobu, Konishi Yuko, Wahiduzzaman Md, Hosokawa Yoshitaka, Konishi Hiroyuki
Department of Biochemistry, Aichi Medical University School of Medicine, Nagakute, Aichi 480-1195, Japan.
Department of Biochemistry, Aichi Medical University School of Medicine, Nagakute, Aichi 480-1195, Japan
Nucleic Acids Res. 2016 Apr 7;44(6):e54. doi: 10.1093/nar/gkv1338. Epub 2015 Dec 10.
The adeno-associated virus (AAV)-based targeting vector has been one of the tools commonly used for genome modification in human cell lines. It allows for relatively efficient gene targeting associated with 1-4-log higher ratios of homologous-to-random integration of targeting vectors (H/R ratios) than plasmid-based targeting vectors, without actively introducing DNA double-strand breaks. In this study, we sought to improve the efficiency of AAV-mediated gene targeting by introducing a 2A-based promoter-trap system into targeting constructs. We generated three distinct AAV-based targeting vectors carrying 2A for promoter trapping, each targeting a GFP-based reporter module incorporated into the genome, PIGA exon 6 or PIGA intron 5. The absolute gene targeting efficiencies and H/R ratios attained using these vectors were assessed in multiple human cell lines and compared with those attained using targeting vectors carrying internal ribosome entry site (IRES) for promoter trapping. We found that the use of 2A for promoter trapping increased absolute gene targeting efficiencies by 3.4-28-fold and H/R ratios by 2-5-fold compared to values obtained with IRES. In CRISPR-Cas9-assisted gene targeting using plasmid-based targeting vectors, the use of 2A did not enhance the H/R ratios but did upregulate the absolute gene targeting efficiencies compared to the use of IRES.
基于腺相关病毒（AAV）的靶向载体一直是人类细胞系中常用于基因组修饰的工具之一。与基于质粒的靶向载体相比，它能实现相对高效的基因靶向，靶向载体的同源整合与随机整合比例（H/R比）高1至4个对数级，且无需主动引入DNA双链断裂。在本研究中，我们试图通过将基于2A的启动子捕获系统引入靶向构建体来提高AAV介导的基因靶向效率。我们构建了三种不同的基于AAV的携带2A用于启动子捕获的靶向载体，每种载体靶向整合到基因组中的基于绿色荧光蛋白（GFP）的报告模块、PIGA外显子6或PIGA内含子5。在多种人类细胞系中评估了使用这些载体获得的绝对基因靶向效率和H/R比，并与使用携带内部核糖体进入位点（IRES）用于启动子捕获的靶向载体所获得的结果进行比较。我们发现，与使用IRES获得的值相比，使用2A进行启动子捕获可使绝对基因靶向效率提高3.4至28倍，H/R比提高2至5倍。在使用基于质粒的靶向载体进行CRISPR-Cas9辅助基因靶向时，与使用IRES相比’使用2A不会提高H/R比，但会上调绝对基因靶向效率。
