Supplementary Information for: Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases by Hockemeyer et al. Nature Biotechnology: doi: 10.1038/nbt.1562Supplemental Figure 1: Surveyor (CEL-I) Nuclease Assay for OCT4 and AAVS1 ZFN pairs A. Screening of ZFNs directed against the human OCT4 locus. The indicated ZFNs (Supplemental Table 1) were transiently transfected into the cell types indicated, genomic DNA was isolated 48 hrs later, and the frequency of target locus disruption was measured by the Surveyor/Cel-1 assay using 32P-labeled PCR exactly as described 26. The frequency of gene disruption is indicated in each gel panel; "G" indicated cells transfected with an eGFP-expression vector as a control. B. BGO1 hESCs were electroporated with an eGFP expression plasmid and the indicated ZFN pairs. As a control cells were electroporated without ZFN. GFP positive cells were isolated by FACS sorting 48 hours after electroporation. DNA was isolated and analyzed for the disruption of the OCT4 and AAVS1 locus. Supplemental Figure 2: FACS analysis of OCT4 targeted clones FACS analysis of BGO1 cells and BGO1 cells targeted with the indicated OCT4 ZFN pairs. All cells were co-stained and analyzed for SSEA4 expression to exclude SSEA4 negative differentiated cells from the analysis. Nature Biotechnology: doi: 10.1038/nbt.1562Supplemental Figure 3: Southern blot analysis of hESCs targeted in the AAVS1 locus A. Representative Southern blot results of BGO1 clones targeted to the AAVS1 locus using the AAVS1-SA-Puro donor plasmid. Southern blot analysis were performed as in Figure 2B. Correctly targeted clones are Nature Biotechnology: doi: 10.1038/nbt.1562indicated in red. Similar to the OCT4 targeting discussed above, a fraction of clones, although targeted, carried additional integrations. The bottom panel shows the analysis of genomic DNA of a selecion of the clones shown above digested with EcoRV and probed with the external 3’ probe. As, EcoRV does not cut in the donor vector the detection of higher than expected molecular weight restriction fragments in clone #3,7,8 and 10 suggests that the additional integration detected with the internal 5’-probe (middle panel) represent multiple integrations of the donor plasmids in the AAVS1 locus rather than an integration caused by off-target effects of the AAVS1 ZNFs. These clones were considered as not correctly targeted and not analyzed further, since the majority of the clones obtained were correctly targeted on one or both ZFN-targeted alleles, and lacked randomly integrated DNA. B. Southern blot analysis as in (A) from targeting experiments with the AAVS1-PGK-Puro donor plasmids. Nature Biotechnology: doi: 10.1038/nbt.1562Supplemental Figure 4: Characterization of BGO1 cells targeted in the AAVS1 locus A. Representative Karyotype analysis of BGO1 cells correctly targeted in the AAVS1 locus using the AAVS1-PGK-Puro donor plasmid. B. Immunofluorescence staining of BG01 cells correctly targeted with the indicated ZFN pairs using the corresponding donor plasmids. Cells were stained for the pluripotency markers OCT4, NANOG, SOX2, Tra-1-60 and SSEA4. C. Hematoxylin and eosin staining of teratoma sections generated from BG01 cells targeted with the indicated ZFN pairs and the corresponding donor plasmids. Nature Biotechnology: doi: 10.1038/nbt.1562Supplemental Figure 5: ZFN mediated gene targeting of hiPSCs A. Southern blot analysis of hiPSC cell line PD21lox-17Puro-5 targeted with the AAVS1 ZFN pairs using the indicated donor plasmids. Genomic DNA was digested with SphI and hybridized with the 32P-labeled external 3’ probe or with the internal 5’ probe. Fragment sizes are: PGK-Puro: 5’-probe: wt=6.5 kb, targeted=4.2 kb; 3’-probe: wt=6.5 kb, targeted=3.7 kb. SA-Puro: 5’-probe: wt=6.5 kb, targeted=3.8 kb; 3’-probe: wt=6.5 kb, targeted=3.7 kb B. Hematoxylin and eosin staining of teratoma sections generated from hiPSC cell line PD21lox17Puro-5 cells targeted with the AAVS1 ZFN pairs using the AAVS1-PGK-Puro donor plasmid. Supplemental Figure 6: Time course experiment of DOX withdrawal of hESCs targeted with a TetO-eGFP cassette in the AAVS1 locus !FACS analysis of BGO1 cells either heterozygous or homozygous for the TetO-eGFP bw donor targeted to the AAVS1 locus. Prior to the experiment cells were transduced with a lentivirus carrying the M2rtTA reverse transactivator, DOX induced and subsequently enriched for eGFP expressing cells by FACS sorting. The graph shows the relative eGFP expression of this cell population at the indicated times after DOX Nature Biotechnology: doi: 10.1038/nbt.1562withdrawal. All cells were co-stained and analyzed for SSEA4 expression to exclude SSEA4 negative feeder cells from the analysis. Supplemental Figure 7: Surveyor (Cel-I) Nuclease Assay for PITX3 ZFNs Two distinct ZFN pairs were tested for disruption of the PITX3 locus in K562 cells as described above. The more active pair (ZFN pair#2) was used for genome editing in hESCs and hiPSCs. Due to a reduced transfection efficiency in hESCs a lower cut frequency than 24.4% of ZFN pair #2 has to be expected in hESCs. Nature Biotechnology: doi: 10.1038/nbt.1562Supplemental Figure 8. Experimental evaluation of genotypes in single-cell derived clones heterozygous for a ZFN-driven targeted integration event at the AAVS1 locus. A. Table of off-target sites identified as detailed in Material and Method section. “FTA” indicates loci that failed to amplify despite extensive PCR optimization and the use of additional primer pairs. Nature Biotechnology: doi: 10.1038/nbt.1562B. A Surveyor endonuclease (Cel-1) assay was performed on genomic DNA from control cells to determine whether a heterozygous SNP or a small indel were present in the region of interest. The lane number corresponds to the number of the putative off-target site in panel A. The black hatch marks on the border of each gel correspond to molecular weight marker positions (100 bp to 600 bp in 100 bp increments). C. Results of Cel-1 assays performed on genomic DNA from 4 randomly chosen genome-edited clones. The number of the off-target site genotyped is indicated above each gel image; the lane number corresponds to the single-cell derived clone being genotyped. Nature Biotechnology: doi: 10.1038/nbt.1562Supplemental Figure 9. Experimental evaluation of genotypes in single-cell derived clones heterozygous for a
View Full Document
Unlocking...