Factors affecting the efficiency of Cas9-mediated viral genome indels during adenovirus infection.

<p>SURVEYOR assay of adenoviral genomes extracted from 293FT cells expressing Cas9:gRNA-175 infected with ADV-EGFP at various time points after transfection (A), using various multiplicities of infection (B), and at different cell harvest time points after viral infection (C). (D) Viral growth curve comparison for recombinant adenovirus-infected 293FT cells expressing the Cas9:gRNA complex or Cas9 protein alone. (E) Schematic showing the F1–F3 regions located in ADV-EGFP viral genomes quantified using qPCR. The Cas9:gRNA-175 target site is indicated. (F) The quantity of the F2 DNA region in infected cells was compared between Cas9:gRNA175-treated ADV-EGFP and the control (Cas9 only). F3 was used to normalize the viral genome content in the different samples, and F1 served as a control. N = 3; error bars show the means ± SEM.