In this study, we developed a single helper-dependent adenovirus (HDAd) to deliver all of the components (donor DNA, CRISPR-associated protein 9 [Cas9], and guide RNA [gRNA]) needed to achieve high-efficiency gene targeting and homology-directed repair in transduced cells. We show that these “all-in-one” HDAds are up to 117-fold more efficient at gene targeting than donor HDAds that do not express CRISPR/Cas9 in human induced pluripotent stem cells (iPSCs). The vast majority (>90%) of targeted recombinants had only one allele targeted, and this was accompanied by high-frequency indel formation in the non-targeted allele at the site of Cas9 cleavage. These indels varied in size and nature, and included large deletions of ∼8 kb. The remaining minority of recombinants had both alleles targeted (so-called bi-allelic targeting). These all-in-one HDAds represent an important platform for accomplishing and expanding the utility of homology-directed repair, especially for difficult-to-transfect cells and for in vivo applications.

在这项研究中，我们开发了一种单一的依赖于辅助基因的腺病毒（HDAd），以提供实现高效基因靶向和靶向所需的所有组件（供体DNA，CRISPR相关蛋白9 [Cas9]和引导RNA [gRNA]）。转导细胞中同源性指导的修复。我们显示，这些“多合一” HDAds在基因靶向方面的效率比在人诱导多能干细胞（iPSCs）中不表达CRISPR / Cas9的供体HDAds高出117倍。绝大多数（> 90％）的靶向重组体只有一个等位基因被靶向，并伴有Cas9切割位点非靶向等位基因中的高频插入缺失形成。这些插入缺失的大小和性质各不相同，并包括约8 kb的大缺失。其余的少数重组体同时具有两个等位基因靶向（所谓的双等位基因靶向）。体内应用。