The CRISPR-Cas9 system is attractive for gene therapy, as it allows for permanent genetic correction. However, as a new technology, Cas9 gene editing in clinical applications faces major challenges, such as safe delivery and gene targeting efficiency. Cas9 is a foreign protein to recipient cells; thus, its expression may prompt the immune system to eliminate gene-edited cells. To overcome these challenges, we have engineered a novel delivery system based on the helper-dependent adenoviral (HD-Ad) vector, which is capable of delivering genes to airway basal stem cells in vivo. Using this system, we demonstrate the successful co-delivery of both CRISPR-Cas9/single-guide RNA and the LacZ reporter or CFTR gene as donor DNA to cultured cells. HD-Ad vector genome integrity is compromised following donor DNA integration, and because the CRISPR-Cas9/single-guide RNA and donor DNA are carried on the same vector, CRISPR-Cas9 expression is concurrently eliminated. Thus, we show the feasibility of site-specific gene targeting with limited Cas9 expression. In addition, we achieved stable CFTR expression and functional correction in cultured cells following successful gene integration.

CRISPR-Cas9系统对基因治疗具有吸引力，因为它可以进行永久性基因校正。然而，作为一项新技术，Cas9基因编辑在临床应用中面临着重大挑战，例如安全递送和基因靶向效率。Cas9是受体细胞的外源蛋白。因此，它的表达可能促使免疫系统消除基因编辑的细胞。为了克服这些挑战，我们设计了一种基于辅助依赖型腺病毒（HD-Ad）载体的新型传递系统，该系统能够在体内将基因传递至气道基底干细胞。使用该系统，我们演示了CRISPR-Cas9 /单向导RNA和LacZ报告基因或CFTR的成功共输送基因作为培养细胞的供体DNA。HD-Ad载体的基因组完整性在供体DNA整合后受损，并且由于CRISPR-Cas9 /单向导RNA和供体DNA携带在同一载体上，因此CRISPR-Cas9的表达被同时消除。因此，我们显示了有限Cas9表达的位点特异性基因靶向的可行性。此外，成功整合基因后，我们在培养的细胞中实现了稳定的CFTR表达和功能校正。