ZFN-driven ORF addition to the PPP1R12C gene (also known as the AAVS1 locus) in various transformed cell types: (A) Schematic of the human PPP1R12C gene (http://www.genome.ucsc.edu), with the exon/intron structure and the ZFN target site indicated. (B) Schematic of donor construct and of the AAVS1 locus following GFP marker ORF addition. The first two exons of the PPP1R12C gene are shown as open boxes. Also annotated are the locations of the splice acceptor site, the 2A ribosome stuttering signal, and a polyadenylation signal (pA). (C) Southern blotting confirms efficient ZFN-dependent ORF addition to the AAVS1 locus in K562 cells. The positions of wild-type and transgenic chromatids are indicated to the right of the gel; the percentage of transgenic chromatids in this cell pool is indicated below lane 2. The PhosphorImager traces used for the quantitation are shown in Supplemental Figure 1. The probe used for Southern blotting, which corresponds to positions chr19:55,628,340–55,628,753 (GRCh37/hg19) is indicated as a purple-filled box; “A” indicates recognition sites for AccI that genomic DNA was cut with for this Southern. (D) Efficient ZFN-driven GFP ORF addition to AAVS1 in K562 cells. Results of a semiquantitative, body-labeled PCR-based assay (see Methods) on cells transfected with the indicated constructs are shown. Primers are located outside of the homology arms and are indicated on the schematic to the right of the gel. The positions of wild-type and transgenic chromatids are indicated to the right of the gel. The frequency of genome-edited chromatids is indicated below each lane. In this assay, when applied to this locus, weak nonspecific incorporation during early PCR cycles produces a band that appears in all samples and migrates above the one generated by the transgenic chromatid. The data below the autoradiograph represent analysis of the frequency of GFP-positive cells by FACS in the same cells genotyped above. (E) As in D, except HEK293T cells were used. (F) As in D, except Hep3B cells were used.

Stability of GFP expression in K562 cells when driven by the endogenous PPP1R12C promoter. Shown is the mean fluorescence intensity of a GFP-positive cell pool (green X's), a clone derived by limiting dilution that is monoallelic at AAVS1 for the GFP ORF (blue squares), diallelic (yellow triangles), and negative control cells (black diamonds) measured over 25 d—∼30 cell doublings—of growth in nonselective medium. After 25 more days of passaging, the MFI remained essentially unchanged (VM Choi and EA Moehle, data not shown).

Gene addition to the AAVS1 locus in hESCs using ZFNs. (A) Schematic overview depicting the editing strategy for both alleles of the PPP1R12C gene. Donor plasmids used to target the locus are shown above; gene elements are represented as in Figure 1B. (Puro) Puromycin resistance gene, (CAGGS) constitutively active CAGGS promoter, (M2rtTA) tetracycline reverse transactivator. (B) Still images from a time lapse movie (see Supplemental movie M1) imaging H2B-eGFP in a representative BGO1 cell targeted with the AAVS1 donor plasmids as shown in A. Scale bar, 10 μm.

Use of ZFNs to generate a panel of U2OS cells carrying glucocorticoid receptor reporter constructs at AAVS1, and their functional characterization. (A) Outline of experiment. (B) Schematic of donor design and of the AAVS1 locus following GFP marker/GRE luciferase reporter addition. Gene elements are represented in the same way as in Figure 1B. (C) Genotype at the AAVS1 locus for clones carrying reporters with GREs derived from genes indicated above each lane. The position of the wild-type (WT) and transgenic (TI) chromatid is indicated to the right of the gel. The “SCNN1AΔ” and “ TSC22D3Δ ” donors have the GR binding site deleted by site directed mutagenesis. Note that the transgenic chromatid is amplified less efficiently than the wild-type one due to a difference in size (see Supplemental Discussion). (D) The single-cell-derived clones genotyped in C were treated with vehicle (EtOH) or dexamethasone (dex); induction of the luciferase reporter was measured and is shown as fold activation by dex over treated with vehicle only.

Use of ZFNs to generate a panel of isogenic HEK293 cells and hESCs carrying distinct shRNA expression cassettes at the AAVS1 locus. (A) Experimental outline. (B) Schematic of donor construct for the HEK293 experiment. Gene elements are represented in the same way as in Figure 1B. shRNA cassettes are driven by a pol III promoter, which is annotated as a white box with an arrow, followed by a red box. (C) Genotypes of the AAVS1 locus (top) and protein expression (bottom) of a panel of single-cell derived HEK293 cell clones. The clones were obtained by FACS and genotyped using primers that lie outside the region of homology with the donor construct (schematic of PCR to the left of the autoradiograph). In all cases, the upper band corresponds to the transgenic, and the lower to the wild-type chromatid, respectively. In a small subset of cases (indicated by asterisks), the clone contains an additional allele of the AAVS1 locus, most likely the result of a DSB-induced deletion. The indicated clones were assayed by Western blot (bottom) for levels of proteins encoded by genes targeted by the indicated shRNAs. A Western blot for a loading control (α-tubulin) is shown at the bottom. (D) Schematic overview depicting the editing strategy for adding shRNA expression cassettes to the AAVS1 locus in hES cells. Annotations are as in Figure 1B. (E) Real-time PCR data (normalized to GAPDH mRNA) for DNMT1 (left) or POU5F1 (right) in pools of single-cell-derived hES clones carrying control shRNAs or three distinct shRNAs directed against DNMT1. See Supplemental Figure 8 for Southern blot clone genotyping data. (F) As in panel E, but with TP53 as the shRNA target. Note that in E and F, for shRNA construct no. 2 for each gene target, a single-cell-derived hES clone was analyzed in this experiment.