Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases

Abstract

Realizing the full potential of human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) requires efficient methods for genetic modification. However, techniques to generate cell type-specific lineage reporters, as well as reliable tools to disrupt, repair or overexpress genes by gene targeting, are inefficient at best and thus are not routinely used. Here we report the highly efficient targeting of three genes in human pluripotent cells using zinc-finger nuclease (ZFN)-mediated genome editing. First, using ZFNs specific for the OCT4 (POU5F1) locus, we generated OCT4-eGFP reporter cells to monitor the pluripotent state of hESCs. Second, we inserted a transgene into the AAVS1 locus to generate a robust drug-inducible overexpression system in hESCs. Finally, we targeted the PITX3 gene, demonstrating that ZFNs can be used to generate reporter cells by targeting non-expressed genes in hESCs and hiPSCs.

Figure 1. Targeting of OCT4 in hESCs using ZFNs

A. Schematic overview depicting the targeting strategy for the OCT4 locus. Probes used for Southern blot analysis are shown as red boxes, exons of the OCT4 locus are shown as blue boxes and arrows indicate the genomic site cut by the respective ZFN pair. Donor plasmids corresponding to the cleavage location of the three ZFN pairs carried 5’ and 3’ homology regions covering roughly 700 bp of the OCT4 sequence flanking the respective DSB target site are shown above; SA-GFP: splice acceptor eGFP sequence, 2A: self-cleaving peptide sequence, PURO: puromycin resistance gene, polyA: polyadenylation sequence. Inset in the upper left depicts a cartoon of two ZFNs binding at a specific genomic site (yellow) leading to the dimerization of the FokI nuclease domains. B. Southern blot analysis of BG01 cells targeted with the indicated ZFN pairs using the corresponding donor plasmids. Genomic DNA was digested either with EcoRI and hybridized with the external 3’-probe or digested with SacI and hybridized with the external 5’-probe or internal eGFP probe. Correctly targeted clones without additional integrations are indicated in red. C. Immunofluorescence staining of BG01 cells 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. D. Hematoxylin and eosin staining of teratoma sections generated from BG01 cells targeted with the indicated ZFN pairs and the corresponding donor plasmids. E. Western blot analysis for the expression of OCT4 and eGFP in BGO1 wild type cells and BG01 cells targeted with the indicated ZFN pairs using the corresponding donor plasmids. Cell extracts were derived from either undifferentiated cells (ES) or in vitro differentiated fibroblast-like cells (Fib.)

Figure 2. Targeting of the AAVS1 locus using ZFNs

A. Schematic overview depicting the targeting strategy for the PPP1R12C gene in the AAVS1 locus. Probes used for Southern blot analysis are shown as red boxes, the first 3 exons of PPP1R12C gene are shown as blue boxes; the arrow indicates the genomic site cut by the AAVS1-ZFNs. Donor plasmids used to target the locus are shown above; SA-Puro: splice acceptor sequence followed by a 2A self-cleaving peptide sequence and the puromycin resistance gene, pA: polyadenylation sequence, PGK: human phophoglycerol kinase promotor, PURO: puromycin resistance gene. B. Southern blot analysis of BG01 cells targeted with the indicated donor plasmids using the AAVS1 ZFNs. 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. C. Southern blot analysis of BG01 cells targeted with an AAVS1 donor plasmid containing a CAGGs driven eGFP cassette using the AAVS1 ZFNs. Genomic DNA was digested with SphI and hybridized with the ³²Plabeled external 3’-probe or with the internal 5’-probe. Fragment sizes are: CAGGs-GFP: 5’-probe: wt=6.5 kb, targeted=3.8 kb; 3’-probe: wt=6.5 kb, targeted=6.9 kb. D. Phase contrast picture and fluorescence imaging of eGFP in heterozygous or homozygous BG01 clones targeted with an AAVS1 donor plasmid containing a CAGGS driven eGFP cassette and the AAVS1 ZFNs. E. Schematic overview depicting the targeting strategy for the PPP1R12C gene in the AAVS1 locus with a donor construct containing a DOXinducible TetO-eGFP. Probes used for Southern blot analysis are shown as red boxes, the first 3 exons of the PPP1R12C gene in the AAVS1 locus are shown as blue boxes and arrows indicate the genomic site cut by the ZFNs. Donor plasmids used to target the AAVS1 locus are shown above; SA-Puro: splice acceptor sequence followed by a 2A self-cleaving peptide sequence and the puromycin resistance gene, pA: polyadenylation sequence, TetO: Tetracycline response element. F. Phase contrast picture and fluorescence imaging of eGFP in BG01 cells either heterozygous (AAVS1 TetO-GFP^+/−) or homozygous (AAVS1-TetOGFP^+/+) for the DOX-inducible eGFP cassette targeted to the AAVS1 locus. Cells were transduced with a M2rtTA lentivirus to render the cells DOX responsive. Panel shows colonies before (top) and after FACS assisted subcloning in the presence of DOX (bottom). G. Southern blot analysis of BG01cells targeted with the indicated donor plasmids using the AAVS1 ZFNs. Genomic DNA was digested with SphI and hybridized with the ³²P-labeled external 3’-probe or with the internal 5’-probe. H. FACS analysis of AAVS1-TetO-GFP^+/− and AAVS1-TetO-GFP^+/+ subclones for GFP expression at different concentrations of DOX. BGO1 cells, targeted cells prior to M2rtTA infection, and subcloned GFP responsive cell lines were cultured at different concentrations of DOX and analyzed. All cells were co-stained and analyzed for SSEA4 expression to exclude SSEA4 negative feeder cells from the analysis.

Figure 3. Targeting of PITX3 in hESCs and hiPSCs using ZFNs

A. Schematic overview depicting the targeting strategy for the PITX3 gene. Probes for Southern blot analysis are shown as red boxes, the first exons of the PITX3 locus are shown as blue boxes and arrows indicate the genomic site cut by the ZFN pair#2. Donor plasmids used to target the PITX3 locus are shown above and contained 5’ and 3’ homologous sequences of approximately 800 bp flanking the predicted ZFN pair #2 target site; eGFP: enhanced green fluorescent protein, PGK: human phophoglycerol kinase promotor, PURO: puromycin resistance gene, loxP: loxP sites, pA: polyadenylation sequence. Two constructs that differed only in the orientation of this selection cassette with respect to the PITX3 gene were successfully used to target PITX3 (See also Table 1). B. Southern blot analysis of BG01 cells targeted with the indicated donor plasmid using the PITX3 ZFNs. The right panel shows Southern blot analysis of clones in which the PGK-Puro cassette was removed by transient Cre-recombinase expression. Genomic DNA was digested with HindIII and probed with ³²P-labeled external 5’-probe or with the internal 3’-probe. Fragment sizes are: 5’ probe: wt=8.8 kb, targeted=7.4 kb Δ-PGK-Puro=10.5 kb; 3’-probe: wt=8.8 kb, targeted=4.3 kb. C. Southern blot analysis of the hiPSCs targeted with the indicated donor plasmids using the PITX3 ZFNs. Genomic DNA was digested with HindIII and probed with ³²P-labeled external 5’ probe or with the internal 3’-probe. Fragment sizes are: 5’ probe: wt=8.8 kb, targeted=7.4 kb; 3’-probe: wt=8.8 kb, targeted=4.3 kb.