Supplementary Figure Legends for Urnov Et Al., Highly Efficient Endogenous Human Gene Correction
Supplementary figure legends for Urnov et al., “Highly Efficient Endogenous Human Gene Correction Using Designed Zinc Finger Nucleases.”
S1. The PCR-based assay for detection of ZFN-induced homology-directed repair is highly quantitative. Indicated percentage of RFLP-carrying DNA was added to wild-type genomic DNA, followed by PCR and digestion with the restriction enzyme corresponding to the RFLP. Percentage of RFLP-derived signal was then determined and compared to the signal frequency expected from the number of RFLP-containing molecules added to each reaction. In this experiment, the square of the correlation coefficient was greater than 0.99.
S2. ZFNs do not indude detectable gross rearrangement of the IL2R locus. a, Restriction map of the IL2R locus. The positions of restriction enzyme sites used to evaluate the integrity of the DNA stretch surrounding exon 5 following ZFN-driven HR is shown. The numbers indicate the size of band expected from a wild-type DNA sequence following Southern blotting with the probe indicated. b, Southern blotting analysis of potential IL2R rearrangements. Genomic DNA transfected with donor DNA only or with donor DNA and the ZFNs (an aliquot of samples 3 and 8 shown in Fig. 3B) was digested with EcoRI alone (lanes 1-2, EcoRI and NcoI (lanes 3-4), EcoRI and ApaI (lanes 5-6), and EcoRI-SphI (lanes 7-8), resolved on a 0.8% agarose gel alongside a 1 kb DNA size marker (Invitrogen), and stained with ethidium bromide to ensure equal DNA loading and visualize the marker. The panel on the right shows the result of Southern blotting performed with a fragment of the X chromosome distal from exon 5 (see panel a). The frequency of HR that introduces a BsrBI site into the middle of exon 5 in each sample is indicated above the autoradiograph in bold lettering. The positions of IL2R-derived fragments are indicated to the right of the gel; additional signal, indicated with an asterisk, is observed in all lanes, most likely due to excess probe used in this experiment to increase sensitivity.
S3. ZFNs do not induce detectable plasmid misintegration. a, Map of the donor DNA plasmid, with positions of the 1.5 kb donor fragment centered on exon 5, and the unique PstI site in the vector backbone indicated. b, Restriction maps of the chromosomal IL2R locus. Top: PstI restriction map, with positions of the donor fragment and IL2R exon 5 relative to the two nearest PstI sites indicated. Bottom: DpnI restriction map of the X chromosome stretch surrounding exon 5, which itself lies in a 650 bp DpnI fragment. c, Results of Southern blotting of IL2R exon 5 following transfection of the following plasmids (left to right): 1, GFP expression plasmid; 2-3 donor DNA plasmid (low and high amount); 4, ZFN expression plasmids; 5-8 both ZFN expression plasmids and donor DNA plasmid (low and high amounts, as indicated). All genomic DNAs analyzed in this Southern blot represented an aliquot of the same sample used for the Southern blot shown in Fig. 3B. The ethidium-bromide stained gel is shown on the left, with positions of the 1 kb marker indicated. On the right is the results of Southern blotting performed with IL2R exon 5. The frequency of ZFN-driven HR that introduced a BsrBI site into the middle of exon 5 (as measured in Fig. 3B) is indicated above the autoradiograph.
S4. ZFNs yield cells homozygous for the genotype of choice. a, Following exposure to ZFNs and donor DNA, cells were seeded to < 1 cell / well, and 30 days later, the exon 5-containing stretch was amplified by PCR, and half the reaction products were resolved on an agarose gel. b, The remaining half were digested with BsrBI and resolved on an agarose gel. c and d, The same experiment was performed, except cells were arrested in G2 for 48 hrs. after transfection with with ZFNs and donor DNA. Genotypes of cells are indicated where appropriate (Bb – 1 allele modified; BB – both alleles modified).
S5. a, K562 cells have lost X chromosome inactivation in the IL2R locus. Genomic DNA was digested with a methylation-sensitive (HpaII) and insensitive (MspI) enzyme and Southern blotting was performed with a probe identifying a CpG island 7 kb upstream of IL2R. b, Single-cell clones of the indicated genotype carry the expected RFLPs in exon 5 of IL2R, as measured by PCR and digestion with the indicated restriction enzymes.
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