Figure 2.

Genome Editing K562 Cells at the CCR5 Locus Requires a Targeted Nuclease

(A) Schematic of the CCR5-PGK-GFP AAV vector genome and the result of HDR genome editing at the CCR5 locus. PGK promoter-driven GFP expression can occur from episomal AAV genomes or following insertion into the host DNA. The positions of in-out PCR primers, to detect site-specific insertion, are indicated. (B) K562 cells were transduced with AAV6, AAV9, or AAV9-G505R vectors packaging CCR5-PGK-GFP genomes at an MOI of 10⁴, and samples were electroporated with CCR5-specific ZFN mRNA as indicated. Flow cytometry plots are shown for indicated treatments and times. (C) Comparison of GFP expression after 14 days in indicated samples, treated with or without ZFN mRNA. (D) Non-quantitative in-out PCR to detect insertion of the PGK-GFP cassette at CCR5. (E) Correlation between cell-associated vector copy number (VCN) at day 2 and GFP expression by flow cytometry at day 14, for indicated samples.