A single “all-in-one” helper-dependent adenovirus to deliver donor DNA and CRISPR/Cas9 for efficient homology-directed repair

Abstract In this study we developed a single helper-dependent adenovirus to deliver all of the components (donor DNA, Cas9 and 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 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.