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

Journal name:
Nature Biotechnology
Volume:
27,
Pages:
851–857
Year published:
DOI:
doi:10.1038/nbt.1562
Received
Accepted
Published online

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.

At a glance

Figures

  1. Targeting of OCT4 in hESCs using ZFNs.
    Figure 1: Targeting of OCT4 in hESCs using ZFNs.

    (a) Schematic overview depicting the targeting strategy for the OCT4 locus. Red boxes, probes used for Southern blot analysis; blue boxes, exons of OCT4; arrows, genomic site cut by the respective ZFN pair. Shown above is a schematic of the donor plasmid design. Donor plasmids were created corresponding to the cleavage location of the three ZFN pairs and carried roughly 700-bp regions of homology to the OCT4 sequence. SA-eGFP, splice acceptor–eGFP sequence; 2A, self-cleaving peptide sequence; PURO, puromycin resistance gene; polyA, polyadenylation sequence. Inset at upper left is a cartoon of two ZFNs binding at a specific genomic site (yellow), leading to 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 either digested 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. Fragment sizes: for 5′ probe and eGFP probe, wt, 6.4 kb, targeted, 8.4 kb; for 3′ probe, wt, 7.1 kb, targeted, 9.1 kb. (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 BG01 wild-type (wt) 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.)

  2. Targeting of the AAVS1 locus using ZFNs.
    Figure 2: Targeting of the AAVS1 locus using ZFNs.

    (a) Schematic overview depicting the targeting strategy for the PPP1R12C gene in the AAVS1 locus. Red boxes, probes used for Southern blot analysis; blue boxes, first 3 exons of PPP1R12C; arrow, 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 promoter; 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 the internal 5′ probe. Fragment sizes for PGK-PURO: 5′ probe: wt, 6.5 kb, targeted, 4.2 kb; 3′ probe: wt, 6.5 kb, targeted, 3.7 kb. Fragment sizes for 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 32P-labeled external 3′ probe or the internal 5′ probe. Fragment sizes for 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 DOX-inducible TetO-eGFP. Red boxes, probes used for Southern blot analysis; blue boxes, first three exons of the PPP1R12C gene in the AAVS1 locus; arrows, 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-TetO-GFP+/+) for the DOX-inducible eGFP cassette targeted to the AAVS1 locus. Cells were transduced with a M2rtTA lentivirus to render them 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 the internal 5′ probe. Fragment sizes: 5′ probe: wt, 6.5 kb, targeted, 3.8 kb; 3′ probe: wt, 6.5 kb, targeted, 5.1 kb. (h) FACS analysis of AAVS1-TetO-GFP+/− and AAVS1-TetO-GFP+/+ subclones for eGFP expression at different concentrations of DOX. BG01 cells, targeted cells before M2rtTA infection, and subcloned DOX-responsive cell lines cultured at different concentrations of DOX were analyzed. All cells were co-stained and analyzed for SSEA4 expression to exclude SSEA4-negative feeder cells from the analysis.

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

    (a) Schematic overview depicting the targeting strategy for the PITX3 gene. Red boxes, probes for Southern blot analysis; blue boxes, first exons of PITX3; arrows, genomic site cut by ZFN pair #2. Donor plasmids used to target the PITX3 locus are shown above; these 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 promoter; 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 PITX3 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. Right, Southern blot analysis of clones in which the PGK-PURO cassette was removed by transient expression of Cre recombinase. Genomic DNA was digested with HindIII and probed with 32P-labeled external 5′ probe or 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 and probed as in b. Fragment sizes: 5′ probe: wt, 8.8 kb, targeted, 7.4 kb; 3′ probe: wt, 8.8 kb, targeted, 4.3 kb.

Accession codes

Referenced accessions

GenBank/EMBL/DDBJ

Author information

  1. These authors contributed equally to this work.

    • Dirk Hockemeyer &
    • Frank Soldner

Affiliations

  1. The Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA.

    • Dirk Hockemeyer,
    • Frank Soldner,
    • Caroline Beard,
    • Qing Gao,
    • Maisam Mitalipova &
    • Rudolf Jaenisch
  2. Sangamo BioSciences, Inc., Richmond, California, USA.

    • Russell C DeKelver,
    • George E Katibah,
    • Ranier Amora,
    • Elizabeth A Boydston,
    • Bryan Zeitler,
    • Xiangdong Meng,
    • Jeffrey C Miller,
    • Lei Zhang,
    • Edward J Rebar,
    • Philip D Gregory &
    • Fyodor D Urnov
  3. Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • Rudolf Jaenisch

Contributions

D.H., F.S. and R.J. designed the experiments and wrote the paper. C.B. provided assistance with construct design and Southern blot analysis. Q.G. analyzed all teratomas. M.M. provided hESCs. J.C.M. and L.Z. designed the ZFNs, which were assembled and tested by R.C.D., G.E.K. and R.A. X.M. performed the SELEX experiments. E.A.B. and B.Z. genotyped ZFN-edited clones for off-target effects. F.D.U., E.J.R. and P.D.G. supervised the ZFN design, characterization and off-target analysis and helped analyze the data and write the paper. D.H. and F.S. performed all other experiments.

Competing financial interests

R.J. is an adviser to Stemgen and a cofounder of Fate Therapeutics. R.C.D., G.E.K., R.A., B.Z., X.M., J.C.M., L.Z., E.J.R., P.D.G. and F.D.U. are full-time employees of Sangamo BioSciences, Inc.

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Supplementary information

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  1. Supplementary Text and Figures (11 MB)

    Supplementary Figures 1–11 and Supplementary Tables 1–3

Additional data