Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan.
Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan; Laboratory of Hepatocyte Regulation, National Institute of Biomedical Innovation, Health and Nutrition, 7-6-8 Saito, Asagi, Ibaraki, Osaka 567-0085, Japan; Global Center for Advanced Medical Engineering and Informatics, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
J Biotechnol. 2019 Oct 10;304:1-9. doi: 10.1016/j.jbiotec.2019.08.004. Epub 2019 Aug 9.
Clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR associated proteins (Cas) 9 system is a powerful tool for genome editing and still being aggressively improved. Cas12a, a recently discovered Cas9 ortholog, is expected to become complementary to Cas9 due to its unique characteristics. Previously we attempted to establish an adenovirus (Ad) vector-mediated delivery of CRISPR-Cas12a system since Ad vector is widely used for gene transfer in basic researches and medical applications. However, we found difficulties preparing of Ad vectors at an adequate titer. In this study, we have developed Ad vectors that conditionally express Cas12a either by a tetracycline-controlled promoter or a hepatocyte specific promoter to avoid putative inhibitory effects of Cas12a. These vectors successfully proliferated in packaging cells, HEK293 cells, and were recovered at high titers. We have also developed packaging cells that express shRNA for Cas12a to suppress expression of Cas12a. Using the cells, the Ad vector directing constitutive expression of Cas12a proliferated efficiently and was successfully recovered at a high titer. Overall, we improved recovery of Ad vectors carrying CRISPR-Cas12a system, thus provided them as a tool in genome editing researches.
成簇规律间隔短回文重复（CRISPR）-CRISPR 相关蛋白（Cas）9 系统是一种强大的基因组编辑工具，并且仍在被积极改进。Cas12a 是一种最近发现的 Cas9 同源物，由于其独特的特性，有望成为 Cas9 的补充。以前，我们试图建立一种腺病毒（Ad）载体介导的 CRISPR-Cas12a 系统的传递，因为 Ad 载体广泛用于基础研究和医学应用中的基因转移。然而，我们发现难以制备足够滴度的 Ad 载体。在这项研究中，我们开发了条件表达 Cas12a 的 Ad 载体，通过四环素控制的启动子或肝细胞特异性启动子表达 Cas12a，以避免 Cas12a 的潜在抑制作用。这些载体在包装细胞、HEK293 细胞中成功增殖，并以高滴度回收。我们还开发了表达 Cas12a 的 shRNA 的包装细胞，以抑制 Cas12a 的表达。使用这些细胞，指导 Cas12a 组成型表达的 Ad 载体有效地增殖，并以高滴度成功回收。总的来说，我们改进了携带 CRISPR-Cas12a 系统的 Ad 载体的回收，从而为基因组编辑研究提供了一种工具。
