Cas9-targeted double genomic loci in recombinant adenoviral genomes.

<p>(A) Schematic of the <i>EGFP</i> gene in a recombinant adenoviral genome with the target sites of gRNA-175 and gRNA-174 indicated by red lines and the corresponding PAM in orange. The expected deletion is highlighted in grey. (B) Electrophoresis of the PCR products, including gRNA-174 and gRNA-175 target sites amplified from recombinant adenoviral genomes. Top: adenoviral genome extracted from 293FT cells expressing Cas9:gRNA and infected with ADV-EGFP (P1). Bottom: adenoviral genome extracted from AD293 cells infected with P1 progeny virus (P2). (C) Example chromatogram showing sequences of double gRNA-mediated (175 and 174) genomic deletions. The junction sites are indicated by red lines. The incidence of each genotype in ten clones is listed in the right-most column.