Bi-allelic Homology-Directed Repair with Helper-Dependent Adenoviruses

Donna J Palmer; Dustin L Turner; Philip Ng

doi:10.1016/j.omtm.2019.10.003

Bi-allelic Homology-Directed Repair with Helper-Dependent Adenoviruses

Mol Ther Methods Clin Dev. 2019 Oct 17:15:285-293. doi: 10.1016/j.omtm.2019.10.003. eCollection 2019 Dec 13.

Authors

Donna J Palmer¹, Dustin L Turner¹, Philip Ng¹

Affiliation

¹ Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.

Abstract

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.

Keywords: CFTR; adenovirus; bi-allelic; cystic fibrosis; gene editing; gene targeting; helper-dependent; homolog-directed repair; homologous recombination.