Palmer Donna J, Turner Dustin L, Ng Philip
Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.
Mol Ther Methods Clin Dev. 2019 Oct 17;15:285-293. doi: 10.1016/j.omtm.2019.10.003. eCollection 2019 Dec 13.
We describe a strategy to achieve footprintless bi-allelic homology-directed repair (HDR) using helper-dependent adenoviruses (HDAds). This approach utilizes two HDAds to deliver the donor DNA. These two HDAds are identical except for their selectable marker. One expresses the puromycin N-acetyltransferase-herpes simplex virus I thymidine kinase fusion gene (PACTk), while the other expresses the hygromycin phosphotransferase-herpes simplex virus I thymidine kinase fusion gene (HyTk). Therefore, puromycin and hygromycin double resistance can be used to select for targeted HDAd integration into both alleles. Subsequently, piggyBac-mediated excision of both PACTk and HyTk will confer resistance to gancyclovir, resulting in footprintless HDR at both alleles. However, gene-targeting frequency was not high enough to achieve simultaneous targeting at both alleles. Instead, sequential targeting, whereby the two alleles were targeted one at a time, was required in order to achieve bi-allelic HDR with HDAd.
我们描述了一种使用辅助依赖型腺病毒（HDAds）实现无足迹双等位基因同源定向修复（HDR）的策略。这种方法利用两种HDAds来递送供体DNA。这两种HDAds除了其选择标记外是相同的。一种表达嘌呤霉素N - 乙酰转移酶 - 单纯疱疹病毒I胸苷激酶融合基因（PACTk），而另一种表达潮霉素磷酸转移酶 - 单纯疱疹病毒I胸苷激酶融合基因（HyTk）。因此，嘌呤霉素和潮霉素双重抗性可用于选择靶向HDAd整合到两个等位基因中。随后，piggyBac介导的PACTk和HyTk的切除将赋予对更昔洛韦的抗性，从而在两个等位基因处实现无足迹HDR。然而，基因靶向频率不够高，无法实现两个等位基因的同时靶向。相反，为了用HDAd实现双等位基因HDR，需要顺序靶向，即一次靶向两个等位基因中的一个。
