Functional genomics, proteomics, and regulatory
DNA analysis in isogenic settings using zinc finger
nuclease-driven transgenesis into a safe harbor
locus in the human genome
Russell C. DeKelver,1Vivian M. Choi,1Erica A. Moehle,1David E. Paschon,1
Dirk Hockemeyer,2Sebastiaan H. Meijsing,3,6Yasemin Sancak,2Xiaoxia Cui,4
Eveline J. Steine,2Jeffrey C. Miller,1Phillip Tam,1Victor V. Bartsevich,1
Xiangdong Meng,1Igor Rupniewski,1Sunita M. Gopalan,1Helena C. Sun,1
Kathleen J. Pitz,1Jeremy M. Rock,1Lei Zhang,1Gregory D. Davis,4Edward J. Rebar,1
Iain M. Cheeseman,2,5Keith R. Yamamoto,3David M. Sabatini,2Rudolf Jaenisch,2,5
Philip D. Gregory,1and Fyodor D. Urnov1,7
1Sangamo BioSciences, Inc., Point Richmond Tech Center, Richmond, California 94804, USA;2The Whitehead Institute for Biomedical
Research, Cambridge, Massachusetts 02142, USA;3Department of Cellular and Molecular Pharmacology, University of California,
San Francisco, California 94158, USA;4Sigma-Aldrich Research Biotechnology, St. Louis, Missouri 63103, USA;5Department
of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA
Isogenic settings are routine in model organisms, yet remain elusive for genetic experiments on human cells. We describe
the use of designed zinc finger nucleases (ZFNs) for efficient transgenesis without drug selection into the PPP1R12C gene,
a ‘‘safe harbor’’ locus known as AAVS1. ZFNs enable targeted transgenesis at a frequency of up to 15% following transient
transfection of both transformed and primary human cells, including fibroblasts and hES cells. When added to this locus,
transgenes such as expression cassettes for shRNAs, small-molecule-responsive cDNA expression cassettes, and reporter
constructs, exhibit consistent expression and sustained function over 50 cell generations. By avoiding random integration
and drug selection, this method allows bona fide isogenic settings for high-throughput functional genomics, proteomics,
and regulatory DNA analysis in essentially any transformed human cell type and in primary cells.
[Supplemental material is available online at http:/ /www.genome.org.]
Transgenesis of human cells is used in functional genomics, pro-
teomics, protein structure-function studies, and cell-based drug
discovery, and is routinely accomplished by random integration
combined with drug selection. Expression of a randomly inte-
grated transgene is unpredictable and tends to be unstable over
time due to epigenetic effects. Further, random integration often
yields multiple integrants per cell, and this can result in the dis-
ruption or activation of host cell genes. Such unintended side ef-
fects produce a non-isogenic experimental setting, and this can
confound data analysis and interpretation.
Work from the Soriano laboratory (Zambrowicz et al. 1997)
has established Rosa26 as the standard locus for transgenesis via
gene targeting (Thomas et al. 1986) in mouse embryonic stem
cells. In the present study, we set out to develop a complementary
classical gene targeting remains challenging due to the relatively
low frequency of correctly targeted events (e.g., Ruis et al. 2008).
We turned to genome editing with designed zinc finger nucleases
(ZFNs) (bottom of Fig. 1A; for review, see Carroll 2008). This tech-
nique emerged from pioneering work by the Jasin, Pabo, Klug, and
Carroll laboratories (Choo and Klug 1994; Rebar and Pabo 1994;
Rouet et al. 1994; Bibikova et al. 2001), and represents a generally
applicable method for the efficient alteration of the genome in
gene disruption in mammalian tissue culture cells (Liu et al. 2010)
andto makean allelicseries ofhistone variantgenesin mousecells
(Goldberg et al. 2010), ZFNs have also been used to drive targeted
gene addition to investigator-specified endogenous loci in human
cells (Lombardo et al. 2007; Moehle et al. 2007; Hockemeyer et al.
2009). ZFNs used for the first editing experiments (Bibikova et al.
2001) relied on three-finger zinc finger proteins and conventional
endonuclease domains; the recent development of methods for
engineering of ZFNs with composite recognition sites of up to 36
bp and carrying high-fidelity endonuclease domains has enabled
highly specific genome editing in both transformed and primary
human cells (Miller et al. 2007; Perez et al. 2008; Hockemeyer et al.
Therefore, we reasoned that ZFNs could be used to enable an
isogenic setting for human cell transgenesis if a suitable genomic
6Present address: Max Planck Institute for Molecular Genetics,
Ihnestrasse 63-73, 14195 Berlin, Germany.
E-mail email@example.com; fax (510) 236-8951.
Article published online before print. Article and publication date are at
online through the Genome Research Open Access option.
20:1133–1142 ? 2010 by Cold Spring Harbor Laboratory Press; ISSN 1088-9051/10; www.genome.org
location could be found. Selection of such a locus to act as a ‘‘safe
that integration of adeno-associated virus (AAV) into the human
is commonly referred to as AAVS1) (Kotin et al. 1992; Tan et al.
2001). AAV infection is not associated with a known pathophysi-
ology (Smithet al. 2008); both hES (Smith et al. 2008; Hockemeyer
et al. 2009) and hiPS (Hockemeyer et al. 2009) cells with a disrup-
tion of PPP1R12C retain pluripotency. Further, this gene is tran-
org) as well as in commonly used transformed cell lines, i.e.,
HEK293, K562, HeLa, DU-145, and Hep3B (RC DeKelver, data not
harbor’’: (1) no known adverse effect on the cell resulting from its
disruption, and (2) transcriptional competence across cell types to
maintain expression from an inserted gene cassette(s).
We describe here a method for using ZFNs to achieve rapid,
efficient transgenesis into the AAVS1 locus in many commonly
used human transformed cell types (K562, HeLa, HEK293, U2OS,
and others) in human fibroblasts and in hES cells. The gene addi-
ZFN/donor-carrying plasmid and produces a pool of cells harbor-
ing the donor-specified novel DNA at the ZFN-specified location.
Both promoterless (i.e., reliant on the native PPP1R12C gene pro-
moter) and promoter-containing inserts placed into the AAVS1
locus exhibit consistent levels of expression over extended pas-
saging in culture.
We describe three applications of this approach by way of il-
lustrating some of the applications of isogenic transgenesis in
hES cell line transheterogygous at the AAVS1 locus for four distinct
genetic entities that together allow inducible gene expression; (2)
we construct a panel of isogenic U2OS cell lines, each carrying
at AAVS1—i.e., in the same chromatin environment—a different
reporter responsive to activation by liganded glucocorticoid re-
ceptor; (3) we generate a panel of isogenic HEK293 cell lines, each
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
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.
ZFN-driven ORF addition to the PPP1R12Cgene(alsoknownastheAAVS1locus)invarioustransformedcelltypes:(A)Schematicofthehuman
1134 Genome Research
DeKelver et al.
carrying at AAVS1 a distinct shRNA expression cassette directed
against a component of the mTOR pathway, and each exhibiting
long-term knockdown of the protein targeted by the small hairpin
RNA (shRNA); we perform the same experiment—but now using
comparably robust target gene mRNA knockdown by the shRNA.
ZFN-driven open reading frame (ORF) addition to the AAVS1
locus: Use in distinct transformed and primary cell types,
and expression stability over time
The PPP1R12C gene is transcribed in all cell types where this issue
has been studied, and this allows the use of promoterless donor
constructs. We designed a panel of ZFNs against nonrepetitive
stretches of its intron 1 using an archive of prevalidated two-finger
modules and screened this panel for endogenous gene disruption
(RC DeKelver and JM Rock, data not shown). The most active ZFN
from the transcription start site of the PPP1R12C gene (Fig. 1A).
The composite ZFN recognition site is unique in the human ge-
nome and is flanked by an extended stretch of single-copy geno-
mic DNA suitable for donor construction.
To trap expression driven by the native PPP1R12C promoter,
the donor construct used (Fig. 1B) contained two 800-bp stretches
of sequence homologous to the region flanking the ZFN site
interrupted by a promoterless green fluorescent protein (GFP) ORF
and a polyadenylation signal. Since exon 1 contains the trans-
lational start site of AAVS1, the donor included a splice acceptor
site, followed by the 2A ribosome stuttering signal (Fang et al.
2005) upstream of the GFP ORF (Fig. 1B). Addition of this cassette
to intron 1 of the PPP1R12C gene yields a single transcript driven
by thenativepromoter (Fig.1B), translationof whichproducesthe
polypeptide encoded by exon 1 of PPP1R12C and, separately, GFP.
Genotyping of K562 cells 48 h following transient trans-
fection with plasmid DNAs encoding the ZFN and donor DNA
construct demonstrated that ;10% of all AAVS1 chromatids in the
cell population had acquired the donor-specified ORF cassette as
gauged by Southern blotting (Fig. 1C; Supplemental Fig. 1) and
a semiquantitative body-labeled PCR assay using primers that an-
neal to the chromosome outside of the region of homology with
the donor (Fig. 1D; see Methods for experimental details). Se-
quencing of the transgenic chromatid from randomly chosen
single-cell-derived clones carrying donor-specified transgenes at
AAVS1 showed that transgenesis occurred in a homology-de-
possibility that in a fraction of cases, gene addition occurs via
a process that uses both homology and end-joining (Richardson
and Jasin 2000), but we have not observed such an outcome using
the AAVS1 ZFNs and plasmid donors delivered to transformed cells
using nucleofection. In agreement with our earlier data on the use
of these and other ZFNs engineered to recognize extended DNA
stretches and carrying high-fidelity FokI domains (Miller et al.
2007; Hockemeyer et al. 2009), a nucleus-wide survey of DSB fre-
quency and measurement of donor plasmid random integration
rates revealed high specificity of ZFN-driven gene addition (Sup-
plemental Figs. 3, 8; Supplemental Discussion).
Control- and ZFN-treated cells were maintained in culture for
1 mo (;35 population doublings) in the absence of selection and
then analyzed by FACS. Consistent with the genotyping data, 13%
pool (Fig. 1D, bottom right). Less than 1% of the cells treated with
the donor plasmid alone expressed GFP (Fig. 1D, bottom left), and
no chromatids transgenic for GFP at AAVS1 were detected in that
sample by Southern blotting or by PCR (lanes 1 in Fig. 1, C and D,
respectively). This showed that ZFN-driven addition of a promo-
terless GFP ORF to the human AAVS1 locus yields ;10% GFP-
positive K562 cells without selection for the desired event.
The same ZFN-donor combination was then used in HEK293
by genotyping (Fig. 1E,F, top) and phenotyping (bottom) were
;3% in the absence of selection and after 1 mo of passaging in
culture. We next tested these ZFNs in a larger panel of transformed
cells along with a donor DNA plasmid with homology arms
flanking a 50-bp heterologous stretch with a novel RFLP (this ar-
rangement allows for accurate measurement of ZFN-driven addi-
tion frequency in a nonradioactive assay) (Supplemental Fig. 4).
With this approach, editing efficiency comparable to that seen in
K562 cells was observed in HCT116 and U2OS cells (Supplemental
observed in A549, DU145, HeLa, HepG2, IMR90, and LNCap cells.
Differences between cell lines in gene-addition frequency could
potentially result from those in ZFN expression levels, in the epi-
genetic state of the AAVS1 locus, and in cell-type-specific bias for
gene addition in primary human cells, we generated integration-
defective lentiviruses carrying the ZFNs and the donor (Lombardo
et al. 2007); infection of hTERT-immortalized human fibroblasts
(Rubio et al. 2002) resulted in ;3% ORF addition to the AAVS1
locus as gauged both by genotyping and phenotyping (Supple-
mental Fig. 5, A and B, respectively).
5, pools of ZFN-edited K562, HEK293, Hep3b cells, and fibroblasts
remained marker positive after 1 mo of continuous passaging in
culture. To more accurately measure the expression stability over
time of a transgene resident in the AAVS1 locus, we used FACS to
isolate GFP-positive K562 cells after transfection with the ZFN-
chromatids in that pool were found to be transgenic for the GFP
ORF (Supplemental Fig. 7B; Supplemental Discussion). Limiting
dilution without additional sorting for GFP expression generated
a panel of single-cell-derived clonal lines exclusively carrying GFP
in a monoallelic or diallelic state (RC DeKelver and EA Moehle,
data not shown). This analysis showed that the ;20% of non-
transgenicchromatids in the GFP-positivepool (Supplemental Fig.
7B) derive from cells with a monoallelic GFP transgene at AAVS1.
Representative control K562 cells, the GFP-positive FACS-enriched
cell pool, and two single-cell clonal lines, monoallelic and diallelic
for a GFP insertion at AAVS1, were grown for 50 cell doublings and
assayed for GFP expression level biweekly. This analysis (Fig. 2)
revealed:(1) noloss of meanfluorescenceintensityoverthe course
of the experiment; (2) consistently higher mean fluorescence in-
tensity of cells diallelic for the GFP cassette at AAVS1 than mono-
allelic (cf. square- and diamond-annotated lines in Fig. 2). These
data showed that ZFN-drivenGFP-ORF additionto the AAVS1 gene
locus results in stable long-term expression of the introduced
transgene in transformed cells.
To demonstrate gene addition to the ‘‘safe harbor’’ in genet-
ically unmodified primary cells, we turned to human embryonic
stem cells (hESCs). DNA delivery to hESCs using electroporation is
inefficient (<5%), and we included selectable markers in donor
we made use of the fact that AAVS1 is autosomal; hence, a euploid
ZFN-driven isogenic transgenesis in human cells
cell provides two distinct cytogenetic locations for transgenes.
Human embryonic stem cells (ESCs) were simultaneously treated
with ZFNs and two distinct donors (Fig. 3A), each carrying a distinct
promoterless selectable marker followed by an expression cassette
for the tet reverse transactivator (Donor A) or histone H2B fused to
agents were genotyped and found to be trans-heterozygous for the
two donor-specified cassettes at the AAVS1 locus (D Hockemeyer,
data not shown). Following doxycyline treatment, uniform intra-
nuclear fluorescence was observed. The cells retained normal hESC
morphology and divided normally (Fig. 3B; Supplemental movie
M1), with the H2B-eGFP fusion protein revealing condensed chro-
mosomes during M phase. In these hESCs, therefore, the AAVS1
locus supported long-term function by four distinct expression
constructs: two promoterless resistance markers driven by the
Taken together, these data showed that (1) ZFNs can be used
range of transformed and primary cell types; (2) the added trans-
gene, when driven by the native PPP1R12C promoter, is expressed
at a stable level over an extended time period in cell culture.
An isogenic panel of U2OS cells carrying glucocorticoid
receptor response element reporter constructs
Next, we set out to determine whether the classical assay in gene
promoter structure-function studies—transient transfection with
reporter constructs—that has been used to study, among other
things, allosteric effects of DNA on function by nuclear hormone
receptors (NHRs) (Meijsing et al. 2009) can be performed at an
isogenic chromosomal location. In this case, integration at the
same genomic locus could allow a direct comparison of reporters
in a defined genomic context with predetermined differences in
the cis elements that orchestrate transcriptional activation by the
glucocorticoid receptor (GR), an extensively studied member of
the NHR superfamily that forms part of a complex response circuit
to corticosteroids (Hager et al. 2009). Using this approach, the
of a GR binding site (GBS) and the presence or absence of cis ele-
ments that functionally interact with the GBS can be assayed. Fur-
thermore, recruitment of GR to such integrated reporters can be
determined by chromatin immunoprecipitation (ChIP), an assay
A panel of donor constructs was assembled (Fig. 4A), each
harboring a distinct GR response element (GRE) of ;1000 bp de-
rived from endogenous GR target genes (Gerber et al. 2009) up-
stream of a basal promoter (FKBP5 and TSC22D3, alias GILZ) or
harboring its own endogenous promoter (SCNN1A, alias ENAC),
driving the expression of a luciferase reporter gene. In all cases,
the donor retains the promoterless GFP cassette, such that if site-
specific gene addition is successful, GFP expression, which is driven
by the native PPP1R12C promoter, would be indicative of the pres-
ence of the reporter construct at the same locus.
U2OS cells expressing GR (Rogatsky et al. 1997) were tran-
siently transfected with AAVS1-targeting ZFNs and each donor
construct; GFP-positive cell pools for each donor were isolated,
single-cell-derived clones produced without drug selection, and
genotyped at the AAVS1 locus. In agreement with data on single-
insert cassettes (Fig. 1), of nine randomly chosen single-cell-de-
rived clones that expressed the luciferase reporter, all nine carried
the desired donor-specified reporter cassette at the AAVS1 locus in
either a monoallelic or diallelic configuration (Fig. 4C). Represen-
tative clones carrying each reporter were then treated with dexa-
methasone, a potent synthetic ligand for the GR. The reporters
recapitulated (Fig. 4D) hormone-dependent activation of the en-
dogenous target genes from which the regulatory sequence was
derived (Gerber et al. 2009) and displayed distinct magnitudes of
transcriptional up-regulation upon ligand treatment (Fig. 4D, cf.
‘‘SCNN1A’’ and ‘‘FKBP5’’ samples). Moreover, ligand-dependent
activation was dependent on the presence of an intact GR binding
site, as deleting the GR binding site of the SCNN1A GRE (Sayegh
et al. 1999) ablated ligand-dependent transcriptional activation
(Fig. 4D, sample ‘‘SCNN1AD’’); the same observation was made
with a line carrying the TSC22D3 promoter lacking GREs (Fig. 4D,
sample ‘‘TSC22D3D’’). Further, hormone-dependent recruitment
of GR to the reporter constructs was observed by chromatin im-
munoprecipitation assays (Supplemental Fig. 6).
Taken together, the data show that the AAVS1 locus can be
used to rapidly construct isogenic panels of cells harboring chro-
mosomal reporters of steroid hormone receptor function that re-
cruit the receptor and exhibit a biologically relevant response to
Isogenic panels of K562, HEK293, and hES cells carrying
functional shRNA cassettes
Next, we set out to determine whether the AAVS1 locus would
allow the long-term function of shRNA expression cassettes. In
buddingyeast, genome-wide reversegenetics experiments are con-
ducted by systematic gene knockout in an isogenic background
(Giaever et al. 2002). The recent development of several whole-
genome collections of expression vectors for shRNAs (Paddison
allowed loss-of-function screens in human cells. These are currently
performed in transient settings or by the random integration of the
shRNA construct via lentivirus transgenesis.
In a pilot study (Supplemental Fig. 7), we used a donor con-
struct carrying promoterless GFP upstream of a U6-promoter-
driven shRNA expression cassette targeting the cell surface marker
endogenous PPP1R12C promoter. Shown is the mean fluorescence in-
tensity 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), dia-
llelic (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).
Stability of GFP expression in K562 cells when driven by the
DeKelver et al.
1136 Genome Research
with AAVS1-targeting ZFNs and this donor, GFP-positive cells
(;8% of total cells; EA Moehle, data not shown) isolated by FACS,
and this cell pool was found to contain ;80% chromatids trans-
genic at AAVS1 (Supplemental Fig. 7B, lane 3; see also Supple-
mental Discussion). To evaluate the efficacy of the inserted shRNA
cassette, we compared control cells and AAVS1 ZFN/donor modi-
fied cells by FACS staining for CD58—the shRNA target molecule
(Supplemental Fig. 7C). Cell-surface staining for CD58 was sig-
nificantly reduced even after 30 cell population doublings (Sup-
plemental Fig. 7C, last sample) and was comparable in magnitude
to that seen 48 h post-transient transfection with the shRNA ex-
pression plasmid itself (Supplemental Fig. 7C, third sample); cells
carrying GFP at the AAVS1 locus were indistinguishable in their
CD58 levels from control cells (Supplemental Fig. 7C).
We next constructed a panel of donor plasmids (Fig. 5B), each
distinct genes in the mammalian target of rapamycin (mTOR)
pathway (Sancak et al. 2008). Plasmids encoding the AAVS1-spe-
cific ZFNs and the individual donor DNA
constructs were transfected into HEK293
cells, and GFP-positive cells isolated by
FACS. Genotyping of each cell pool re-
vealed significant single-step enrichment
Western blotting of the pool of GFP-
positive cells was performed to assess pro-
tein levels for the shRNA-targeted gene
products. For two of the three shRNA tar-
gets studied (TSC2 and RPTOR) we ob-
served lower target protein levels than
control cells (Y Sancak and DM Sabatini,
data not shown). This was not the case
for either of the RRAGC-targeting shRNA
constructs we tested, despite comparable
transgenesis levels. We next isolated and
genotyped a panel of GFP-positive single-
cell-derived clones for each shRNA and
identified clonal cell lines monoallelic
and diallelic for gene addition at AAVS1
(Fig. 5C, top). In the case of all three gene
targets, single-cell-derived clones carry-
ing diallelic gene addition of the relevant
shRNA cassette exhibited a significant
knockdown of the target protein level
(Fig. 5C, bottom). Of note, ;8 wk of con-
tinued passaging had elapsed since the
cells were initially treated with the ZFNs
and the donor cassette. Since ZFN-driven
gene addition occurs within 24–48 h of
transient transfection, these data indi-
cate that the AAVS1 locus provides a
suitable genomic environment for long-
term function by shRNA expression cas-
In earlier work (Hockemeyer et al.
2009) and in Figure 3 we have demon-
strated the feasibility of ZFN-driven ad-
dition of transgenes carrying RNA pol II
promotersto AAVS1in hiPSandhEScells.
We next asked whether the AAVS1 locus
could function as a safe harbor for shRNA expression cassettes in
hESCs. Single-cell-derived hESC clones carrying shRNAs directed
against TP53, DNMT1, or control shRNAs were generated using
ZFNs and donor constructs (Fig. 5D). In agreement with nucleus-
wide data from transformed cells (Supplemental Fig. 3), Southern
blotting showed that >90% of hESC clones that carry the desired
transgene at AAVS1 lack additional random donor integrants
(Supplemental Fig. 8; Supplemental Discussion). Clones were ex-
panded, mRNA isolated, and expression levels of the genes tar-
geted by the shRNAs measured (Fig. 5E,F). In the case of both
genes, single-cell-derived clones carrying target-specific, but not
control shRNA expression cassettes, exhibited stable long-term
knockdown of mRNA levels (Fig. 5E,F); importantly, hESC clones
carrying DNMT1-targeted shRNAs transcribed POU5F1, a key
marker of pluripotency, to the same level as control cells (Fig. 5E,
driven ‘‘safe harbor’’ gene addition process to rapidly, and in some
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 mm.
Gene addition to the AAVS1 locus in hESCs using ZFNs. (A) Schematic overview depicting
ZFN-driven isogenic transgenesis in human cells
cases without drug selection, obtain a panel of isogenic cells car-
rying investigator-specified shRNAs at a defined location, and
exhibiting robust knockdown of the mRNA (Fig. 5E) or protein
(Fig. 5C) encoded by the gene that the shRNA targets.
Isogenic settings are standard in experimental systems such as
budding yeast, Drosophila, and laboratory mice, where they can be
achieved via well-established protocols (e.g., see Osborne et al.
2009). The work we describe adds transgenesis in human trans-
formed andprimary cells to the list of such systems.Gene addition
occurs in a single step within 48 h of transient transfection, relies
on plasmid DNA constructs, functions across a range of trans-
fibroblasts and hES cells (this work) and in induced pluripotent
native PPP1R12C gene promoter, as well as carrying their own pol
II and pol III promoters, function when placed into the AAVS1
locus over extended passaging in culture. The method we describe
provides a complement to other techniques available for trans-
gene placement into the same location in human cells, includ-
ing two-step recombination-based cassette exchange (Sauer
and Henderson 1988) and gene targeting with recombinant AAV
vectors (Kohli et al. 2004). The frequency of the addition process
observed with ZFNs often obviates the need for a drug selection
step on the path to obtaining the cells of interest, which is re-
quired with other approaches. In further contrast, the use of
ZFNs and donors carrying promoterless markers allows the rapid,
single-step isolation of a cell pool or single-cell-derived clones
that carry the marker-linked transgene at the desired location
(e.g., Figs. 4, 5).
Earlier data on gene targeting (Smith et al. 2008) or ZFN-
driven gene addition to AAVS1 (Hockemeyer et al. 2009) showed
that hES and iPS cells disrupted at that locus retain pluripotency.
Further, ZFN-edited human ES cells carrying transgenes at the
AAVS1 locus retained a normal karyotype, expression of pluri-
potency markers, and remained pluripotent as well as wild type at
a panel of putative off-target sites (Hockemeyer et al. 2009). In our
experiments, K562, Hep3B, HEK293, U2OS, fibroblast, and hES
cells carrying transgenes at AAVS1 proliferated indistinguishably
from control cells; the hES cells shown growing and dividing
normally in Supplemental movie 1, for instance, carry PPP1R12C-
disrupting transgenes at both its alleles. This said, it remains for-
mally possible that some pathway may be adversely affected by
is thought to regulate actomyosin-based contractility) (Mulder
et al. 2004). If such a pathway is found, which could be attempted,
for instance, by genome-wide expression analysis of edited cells or
by knocking out the orthologous gene in mouse or in rat (Geurts
et al. 2009), we have shown that single-cell-derived clones pos-
sessing one wild-type and one transgenic AAVS1 allele can be
readily isolated (e.g., see Figs. 2, 4, 5) (in a typical experiment,
60%–70% of edited cells fall into that category). We are unaware of
evidence that PPP1R12C is haploinsufficient.
We describe three applications for isogenic transgenesis into
a ‘‘safe harbor.’’ We show that four distinct coding regions—two
site specifically in allelic positions at the AAVS1 locus to generate an
inducible expression system (Fig. 3). In the present work, we de-
scribe hES cells carrying a histone H2B-eGFP fusion in such an in-
ducible setting. Studies by Grunstein and colleagues (Kayne et al.
1988) launched an ever-growing line of genetic investigation into
histone structure and function; the system we describe allows the
acterization. (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 ‘‘SCNN1AD’’
and ‘‘ TSC22D3D ’’ 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
Use of ZFNs to generate a panel of U2OS cells carrying glucocorticoid receptor reporter constructs at AAVS1, and their functional char-
DeKelver et al.
1138 Genome Research
rapid construction of a panel of isogenic cells carrying, for in-
stance, an allelic series of histone genes (see also Goldberg et al.
2010). Moregenerally, high-throughput proteomic (e.g.,structure-
that is normalized across different cell lines for expression of the
transgene and its integration site in the genome. The native
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, andthe lower to thewild-type chromatid, respectively. Inasmall subset ofcases(indicated by asterisks), the clonecontains anadditional
alleleofthe AAVS1 locus, most likelythe result ofaDSB-induced deletion. Theindicated cloneswere assayed byWestern blot (bottom)for levels ofproteins
encoded by genes targeted by the indicated shRNAs. A Western blot for a loading control (a-tubulin) is shown at the bottom. (D) Schematic overview
(normalized to GAPDH mRNA) for DNMT1 (left) or POU5F1 (right) in pools of single-cell-derived hES clones carrying control shRNAs or three distinct
shRNAsdirectedagainstDNMT1.SeeSupplementalFigure 8forSouthernblotclonegenotypingdata.(F )AsinpanelE,butwithTP53astheshRNAtarget.
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.
Use of ZFNs to generate a panel of isogenic HEK293 cells and hESCs carrying distinct shRNA expression cassettes at the AAVS1 locus. (A)
ZFN-driven isogenic transgenesis in human cells
PPP1R12C promoter is active in all cells where we and others have
studied this issue; in our hands, K562 and HeLa cells carrying pro-
moterless GFP transgenes at AAVS1 show lower mean fluorescence
PGK promoter (VM Choi and EA Moehle, data not shown). While
weaker by this criterion, the native PPP1R12C promoter drives
sufficient transcription to allow FACS or selection-based isolation
of pools and clones of cells with correctly added promoterless
markers (e.g., Figs. 2–5).
Wealsoshowthat theAAVS1 locuscanbe usedtocarrya panel
of reporter constructs for the study of transcriptional cis-regulatory
elements (Fig. 4). In the wake of initial studies in the 1980’s
accumulated that connects targeted chromatin remodeling and
modification to actionby nuclear hormone receptors (Stallcupetal.
2003; Hager et al. 2009) and most transcription factors where this
issue has been studied. The ability to place investigator-specified
reporters into the AAVS1 locus offers an opportunity to study ge-
pathways; preliminary studies (SH Meijsing, unpubl.) show the re-
porter transgenes exhibit kinetics of induction by hormone and
Finally, we demonstrate that the AAVS1 locus serves as a safe
(Fig. 5). Functional genomics using RNAi is an important research
tool and is commonly performed in transient settings where RNAi
hairpins or transient transfection approaches suffice. The data we
show demonstrate the feasibility of rapidly obtaining single-cell-
derived clones isogenic except for a construct expressing a shRNA
hairpin against the gene of choice. Robust target gene knockdown
is still observed 2 mo after transgenesis. In transformedcells, linear
donors with short homology arms produced by PCR may be used
(Orlando et al. 2010), or ZFNs and a donor construct can be com-
bined onto the same plasmid without significant loss of efficiency
(Supplemental Fig. 9), illustrating two simple paths to establishing
genome-wide screening with shRNA constructs stably integrated
into the AAVS1 locus.
These data, therefore, expand the toolbox of human somatic
cell genetics to include transgenesis in isogenic settings.
Zinc finger nucleases and donor constructs
ZFNs against the AAVS1 locus (Hockemeyer et al. 2009) were used
in their obligate heterodimer, high-fidelity FokI (Miller et al. 2007)
form cloned into pVAX as a 2A fusion construct (Perez et al. 2008).
Donor constructs for RFLP addition and gene addition were as-
see also below); shRNA expression constructs for the experiment
shown in Figure 5A were obtained from Sigma-Aldrich.
Cell culture techniques for K562, HeLa, and HEK293 cells (Moehle
et al. 2007), U2OS cells (Meijsing et al. 2009), hTERT fibroblasts
(Lombardo et al. 2007), and hESCs (Hockemeyer et al. 2008) have
been described previously. All other cell types were cultured as per
Genome editing in transformed cells and in fibroblasts
ZFN expression and donor constructs, assembled using standard
recombinant DNA techniques (detailed protocols are available
from the authors upon request) were introduced into transformed
cells by nucleofection (Amaxa) exactly as described (Urnov et al.
2005; Moehle et al. 2007) and into hTERT fibroblasts using
integration-defective lentivirus generated exactly as described
(Lombardo et al. 2007). Genomic DNA was harvested using
QuickExtract (Epicentre) for PCR-based assays and DNEasy (Qia-
gen) for Southern blotting performed exactly as described (Urnov
et al. 2005; Moehle et al. 2007); for the Southern blot shown in
Figure 1, genomic DNA was digested with AccI. Targeted integra-
tion frequency was measured using a body-labeled PCR assay as
described (Moehle et al. 2007) with minor modifications. One
hundred nanograms of genomic DNA was amplified (forward
ACCCCGAAGAGTG—both anneal to the chromosome outside
the region of homology with the donor) in the presence of 5 mCi
each of [a-32P]dCTP and [a-32P]dATP using Accuprime HiFi Taq
polymerase (Invitrogen) and the following PCR settings: 95°C,
3 min; 24 cycles of 95°C, 30 sec; 62°C, 30 sec; 68°C, 4 min; 68°C,
a column-based kit (Qiagen) and resolved on a 5% nondenaturing
PAGE; the gel was dried and analyzed using a PhosphorImager
(Molecular Dynamics). Cells were phenotyped for fluorescence
using a Guava benchtop flow cytometer exactly as described
(Moehle et al. 2007).
Genome editing in BGO1 hESCs using ZFNs
hESCs were cultured in Rho Kinase (ROCK)-inhibitor (Calbio-
chem; Y-27632) 24 h prior to electroporation. Cell were harvested
using 0.25% trypsin/EDTA solution (Invitrogen) and 1 3 107cells
resuspended in phosphate buffered saline (PBS) were electro-
porated with a total of 40 mg of donor plasmids (designed and as-
sembled by D Hockemeyer) and 5 mg of the AAVS1 ZFN encoding
plasmid using a Gene Pulser Xcell System, Bio-Rad: 250 V, 500 mF,
0.4 cm cuvettes (Costa et al. 2007). Cells were subsequently plated
on MEF feeder layers DR4 MEFs in hESC medium supplemented
with ROCK-inhibitor for the first 24 h. Individual colonies were
picked after puromycin selection (0.4 mg/mL) and neomycin se-
lection (50 ng/mL) and expanded 10–14 d after electroporation.
used to identify hESC clones with unique integrations of each
donor plasmid in one of the AAVS1 alleles.
Live cell imaging
Targeted hESCs (BG01 [NIH Code: BG01; BresaGen, Inc.]) were
cultured for the duration of imaging on mitomycin C inactivated
dishes (MatTek Corporation) in CO2independent medium (Gibco)
supplemented with 15% fetal bovine serum (FBS) (Hyclone), 5%
KnockOut Serum Replacement (Invitrogen), 1 mM glutamine
(Invitrogen), 1% nonessential amino acids (Invitrogen), 0.1 mM
b-mercaptoethanol (Sigma), and 4 ng/mL FGF2 (R&D systems).
Cells were cultured and maintained in the presence of 2 mg/mL
doxycycline for more than 2 wk prior to imaging to induce and
maintain H2B-eGFP expression. Cells were imaged over a period of
20 h using a Detavision microscope (403 objective) and a HQ2
camera. Time lap intervals were 4 min, and seven 1.5 mm Z-stack
images were taken for each time point with an exposure time of
0.25 sec and 10% transmittance.
Luciferase reporter assays and Western blotting
For reporter activity assays, single-cell derived clonal lines were
seeded into 24-well plates in DMEM/5% FBS at ;20,000 cells per
DeKelver et al.
well and treated the next day with either vehicle (ethanol) or
100 nM dexamethasone. After treatment for ;13 h, cells were lysed
in 100 mL per well of 13 lysis buffer (PharMingen) and assayed for
luciferase activity, which was normalized for cell number. Western
blotting was performed as described (Sancak et al. 2008).
Reverse transcription of total RNA and real-time PCR
RNA was isolated from hESCs, which were mechanically separated
from feeder cells using TRIzol extraction and subsequent pre-
cipitation. Reverse transcription was performed on 1 mg of total
at 50°C(Invitrogen).Real-timePCRwasperformedin anABIPrism
7900 (Applied Biosystems) with Platinum SYBR green pPCR
SuperMIX-UDG with ROX (Invitrogen). Primers for the analysis of
endogenous gene expressions were: hrtdnmt1_F ggttcagcaaaaccaa
tctatgatg; hrtdnmt1_R gccaagatttttgccattaacac; hrtoct4_F gctcgaga
aggatgtggtcc; hrtoct4_R cgttgtgcatagtcgctgct; hrtp53_F gcccccaggg
aggagcacta; hrtp53_R gggagaggagctggtgttg (the latter two from
Boley et al. 2000). Gene expression was normalized using GAPDH
primers: hrtgapdh_F cagtcttctgggtggcagtga; hrtgapdh_R cgtggaagg
We thankJudith Campisifor the gracious gift of hTERT fibroblasts,
William Hahn for proposing the experiment that resulted in the
datashown inFigure5, EdwardWeinsteinforshRNAreagents,and
Edda Einfeldt for technical assistance. We deeply appreciate sug-
and thank the three anonymous referees for comments on the
manuscript. D.H. is a Merck Fellow of the Life Science Research
Foundation.R.J. was supportedby US NationalInstitutesof Health
grants R37-CA084198, RO1-CA087869, and RO1-HD045022 and
by the Howard Hughes Medical Institute. R.J. is an adviser to
Stemgen and a cofounder of Fate Therapeutics. S.H.M. and K.R.Y.
received research support from NIH grants, K.R.Y. is a paid con-
sultant with Merck and Company. Y.S. and D.M.S. were supported
of Defense (W81XWH-07-0448), and W.M. Keck Foundation.
D.E.P., J.C.M., X.M., L.Z., E.J.R., P.D.G., and F.D.U. are full-time
current, and R.D., V.M.C., E.A.M., I.R., S.M.G., H.C.S., K.J.P., and
J.M.R. are past employees of Sangamo BioSciences, which has filed
a patent application based on this work; X.C. and G.D.D. are full-
time current employees of Sigma-Aldrich Biotechnology.
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Received February 18, 2010; accepted in revised form May 11, 2010.
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