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DNA-binding Specificity Is a Major Determinant of the Activity and Toxicity of Zinc-finger Nucleases
original article
© The American Society of Gene Therapy
DNA-binding Specificity Is a Major Determinant of the Activity and Toxicity of Zinc-finger Nucleases Tatjana I Cornu1, Stacey Thibodeau-Beganny2, Eva Guhl1, Stephen Alwin1, Magdalena Eichtinger2,3, JK Joung2,3 and Toni Cathomen1 1 Charité Medical School, Institute of Virology (CBF), Berlin, Germany; 2Molecular Pathology Unit, Center for Cancer Research, and Center for omputational and Integrative Biology, Massachusetts General Hospital, Charlestown, Massachusetts, USA; 3Department of Pathology, C Harvard Medical School, Boston, Massachusetts, USA
The engineering of proteins to manipulate cellular genomes has developed into a promising technology for biomedical research, including gene therapy. In particular, zinc-finger nucleases (ZFNs), which consist of a nonspecific endonuclease domain tethered to a tailored zinc-finger (ZF) DNA-binding domain, have proven invaluable for stimulating homology-directed gene repair in a variety of cell types. However, previous studies demonstrated that ZFNs could be associated with significant cytotoxicity due to cleavage at off-target sites. Here, we compared the in vitro affinities and specificities of nine ZF DNA-binding domains with their performance as ZFNs in human cells. The results of our cell-based assays reveal that the DNA-binding specificity—in addition to the affinity—is a major determinant of ZFN activity and is inversely correlated with ZFN-associated toxicity. In addition, our data provide the first evidence that engineering strategies, which account for context-dependent DNAbinding effects, yield ZFs that function as highly efficient ZFNs in human cells. Received 1 September 2007; accepted 15 October 2007; published online 20 November 2007. doi:10.1038/sj.mt.6300357
Introduction Engineered proteins that permit the targeted modulation of a cellular genome represent an important tool for genetic studies, biotechnology, and gene therapy.1–5 Zinc-finger nucleases (ZFNs) are customizable artificial nucleases composed of an engineered zinc-finger (ZF) domain linked to the cleavage domain of the type IIs restriction endonuclease FokI.6,7 ZFNs can be engineered to bind and cleave chromosomes at a desired locus and are therefore invaluable for stimulating site-specific genetic modifications based on homologous recombination (HR). HR is a rare event in mammalian cells,8 but the frequency can be dramatically improved by the introduction of a targeted DNA double-strand break (DSB) near the site of the desired recombination event.9,10 Several studies have reported gene targeting frequencies between 1 and 20% when a DSB was introduced by engineered ZFNs.11–19
To generate a ZFN directed against a specific DNA target sequence, a DNA-binding domain has to be engineered that contains three or four artificial ZF domains. Such ZFs are small domains of ~30 residues, which consist of a ββα-fold coordinated by a zinc ion. The structure of the transcription factor Zif268, which contains three ZF domains, revealed that each finger uses residues located at the amino-terminal end of its α-helix to specifically bind three to four bases of DNA.20,21 A large number of studies have shown that alteration of six amino acids in the αhelix is sufficient to create ZFs with novel DNA-binding specificities. Using a combination of rational design and/or selection strategies, a library of ZF modules has been created that includes DNA-binding domains able to recognize most of the 64 possible triplets.22–30 Individual ZFs can be assembled into tandem arrays, which can recognize a wide variety of novel target sites. Because ZFNs cleave DNA as dimers, two ZFN subunits are typically designed to recognize the target sequence in a tail-to-tail conformation.31,7 The C-terminal FokI domains are positioned in close proximity on the same face of the DNA helix by separating the two ZFN binding sites by a 5- or 6-base pair (bp) spacer.32 Previously published studies have reported that not all engineered ZF domains exhibit optimal behavior as ZFNs. While some of these ZFNs lack activity at all others induce significant cytotoxicity in human cells, an effect proposed to be caused by cleavage at “off-target” sites.12,13,17–19 ZFN-induced toxicity is likely determined by multiple factors, including the specificity of DNA binding and DNA cleavage as well as the frequency and location of sites in the genome actually bound by the ZFNs. We and others recently described a structure-based approach to reducing ZFN-induced toxicity.18,19 Rational redesign of the FokI domain was used to create an asymmetric dimer interface that destabilized dimerization and forced heterodimerization of the ZFN subunits.19 The resulting ZFNs elicited significantly reduced cytotoxicity without compromising on performance, suggesting that architectural changes in the ZFN structure can improve the specificity of DNA cleavage.18,19 Another study presented data suggesting that cytotoxicity could be reduced by identifying ZFN variants with “improved in vitro DNA-binding and cleavage properties”.14 However, the authors of this report did not specify the nature of the optimization performed.
Correspondence: Toni Cathomen, Charité Medical School, Institute of Virology (CBF), Hindenburgdamm 27, D-12203 Berlin, Germany. E-mail: [email protected]
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Specificity, Activity, and Toxicity of ZFN
In this study, we sought to understand the relationship between the DNA-binding affinities and specificities of ZF domains and their performance and toxicity as ZFNs in human cells. To this end, we examined various ZFNs engineered to bind to the same sites but which possess different DNA-binding affinities and specificities. Our results demonstrate that for a given DNA target site, both the DNA-binding affinity and specificity of the ZF domain influence the activity and toxicity of a ZFN. Our data also provide the first evidence that ZF domains engineered using a bacterialtwo-hybrid (B2H)-based selection strategy function well as ZFNs in human cells.
Results Evaluation of engineered ZF domains using transcriptional activation assays To assess the relationship between the in vitro DNA-binding affinity and specificity of engineered ZF domains and their performance as ZFNs in human cells, we chose a series of nine
a ZF
Target site
Zinc-finger sequences (–1 to +6)
KdS mol/l
KdN nmol/l
Specificity
EB0 5′EB1 EB2
150 31 65
1,000 1,100 1,100
6,700 35,000 17,000
HV0 5′HV1 HV2
– 9 9
– 1,100 180
– 87,000 19,000
BA0 5′BA1 BA2
28 78 60
55 2,100 1,300
1,980 27,000 23,000
AD
b 3
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Mammalia 30
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*
25
*
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*
*
G11
c 3
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cto
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5 0
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Molecular Therapy vol. 16 no. 2 feb. 2008
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Fold-activation
20
previously described ZF domains directed against target sites in the erbB2 locus (EB), a BCR-ABL translocation sequence (BA), or the human immunodeficiency virus-1 promoter (HV).33 For each of the three DNA target sequences three ZF domains were generated, each possessing varying affinities and specificities for their binding sites (Figure 1a). We initially characterized these nine ZF domains using two different transcriptional reporter assays designed to serve as surrogate assays of DNA binding in cells: one based in human cells and the other in bacteria. For the human cell-based assay, we constructed expression vectors encoding each of the nine different ZF domains fused to the VP16 transcriptional activation domain to create artificial transcription factors. Each of these ZF-activatorencoding plasmids were transiently transfected into human 293T cells together with a luciferase reporter bearing either a cognate or non-cognate upstream binding site for the ZF to be evaluated (Figure 1b). As expected based on the in vitro DNA-binding data, the EB1 and EB2 activators stimulated transcription from their specific reporter more efficiently than the EB0 activator, and the HV1 and HV2 activators performed better than the HV0 activator. Surprisingly, the BA0 activator stimulated luciferase expression more efficiently than either the BA1 or BA2 activators. These results suggest that within a set of ZF domains targeted to a given target site, transcriptional activation in the mammalian cellbased assay correlates well with the in vitro DNA-binding affinity (Figure 1a) of the ZF domain tested. To analyze the ZF domains in a B2H assay, the ZFs of interest were fused to a fragment of the yeast Gal11P protein and expressed in bacterial cells harboring a single-copy reporter vector. The reporter bears the cognate ZF target sequence upstream of a weak promoter, which directs expression of a lacZ reporter gene (Figure 1c). These reporter cells also express a second hybrid protein consisting of an RNA polymerase α-subunit domain fused Figure 1 Characteristics of engineered zinc-finger (ZF) domains. (a) In vitro data. The genomic DNA target sites and the sequences of the crucial residues in the recognition helix (positions −1 to +6) are indicated for each ZF module. The affinities and specificities have been determined previously.33 KdS, affinity to target site; KdN, affinity to nonspecific site; the specificity is the ratio of KdN/KdS. (b) Mammalian cellbased assay. For expression in human cells, the VP16 transcriptional activation domain (AD) is fused to the SV40 nuclear localization signal (black oval), and three ZF modules (1, 2, 3). The reporter plasmid contains the luciferase gene downstream of a minimal herpes simplex virus thymidine kinase promoter element (TATA), and upstream binding sites for the ZFs (grey boxes, each box represents 3 base pair of the target site). 293T cells were transfected with expression plasmids for the custom AD-ZF, reporter plasmids containing either two binding sites for the EB fingers (EB/EB) or a combined target site for the HV and BA fingers (HV/BA). Luciferase activity was normalized for transfection efficiency and is shown relative to transfection with empty vector (cto). (c) Bacterial cell-based assay. In the bacterial-two-hybrid (B2H) reporter system, expression of the lacZ reporter gene is dependent upon binding of the ZF–Gal11P (G11) hybrid protein to the target site. ZF–G11 interacts with the Gal4 (G4)-RNAP α-subunit (Rα) hybrid protein, which then recruits the RNA polymerase to the weak downstream promoter (−35/−10). The B2H reporter strains were doubly transformed with plasmids expressing the ZF–G11 and G4–Rα hybrid proteins and transformants were assayed for β-galactosidase activity. Fold-activation of lacZ expression is shown relative to transfection with empty vector (cto). Statistical significance for both assays is indicated by * (P < 0.007) and ** (P < 0.001).
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Specificity, Activity, and Toxicity of ZFN
to a fragment of the yeast Gal4 protein, known to interact with Gal11P. In contrast to the results obtained with the mammalian system, the BA0 domain activated transcription less efficiently than the BA1 or BA2-based activators, suggesting that the DNAbinding specificity—in addition to affinity—is a determinant of the activity of a ZF domain in the B2H system.34,33
DNA-binding specificity contributes to ZFN activity in human cells ZFN expression plasmids were generated by fusing each of the nine ZF domains to the cleavage domain of the FokI endonuclease (Figure 2a). Western blot analysis confirmed that all ZFNs were expressed at comparable steady-state levels in human cells (Figure 2b). A recently described recombination assay13 was used to quantify the activities of these nine ZFNs in human cells by measuring their abilities to stimulate HR between a target plasmid and a repair plasmid (Figure 2c). The target plasmid harbors a lacZ gene followed by stop codons and a 5′-truncated
a
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% GFP-positive cells
d 60
Figure 2 Stimulation of episomal gene targeting by designer zincfinger nucleases (ZFNs). (a) Design of ZFNs. All ZFNs contain an Nterminal HA tag (small oval), followed by three ZF modules (1, 2, 3), a short AAARA linker and the catalytic domain of FokI. (b) ZFN expression levels. Transfected 293T cells were harvested after 30 hours and lysates probed with an antibody against the HA tag. “cto” indicates transfection with a control vector. Size markers are indicated on the left. (c) Experimental set up. Repair plasmid (RP) and target plasmid (TP) are described in the text. The recognition site for I-SceI and the homodimeric binding sites for the EB-based ZFNs are highlighted. (d) Episomal gene targeting. 293T cells were transfected with RP, TP and nuclease expression vectors. The columns designate the percentage of enhanced green fluorescent protein (EGFP)-positive cells 2 days post-transfection as determined by flow cytometry. CMV, cytomegalovirus. Promoter; SV40, Simian virus 40 promoter.
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enhanced green fluorescent protein (EGFP) gene (∂GFP), which ensures that no functional EGFP is expressed. The target sites for each of the three sets of ZFNs (EB, HV, BA) were placed as inverted repeats of the respective 9-bp binding sites between the lacZ and truncated EGFP open reading frames together with the cleavage site for the meganuclease I-SceI (as a control). The repair plasmid harbors a non-transcribed 5′-truncated lacZEGFP fusion gene and is designed to rescue EGFP expression through HR by generating a lacZ-EGFP fusion gene. For all target loci, only a few cells turned green in the absence of an expressed nuclease (cto), representing non-stimulated gene targeting (Figure 2d). Expression of EB0-based ZFN, EBO, stimulated HR only poorly, whereas expression of EB1-N and EB2-N stimulated gene correction to a similar extent as I-SceI, with some 40% of transfected cells expressing a functional lacZ-EGFP gene. (Note that 40% of EGFP-positive cells does not correspond to conversion of 40% of the target DNAs because each cell contains many plasmids.) Similarly, expression of HV1-N and HV2N as well as BA1-N and BA2-N stimulated HR to a larger extent than HV0-N and BA0-N, respectively. We then assessed performance of the nine different ZFNs using a chromosomal version of our HR assay. To do this, we generated specifically designed target cell lines, in which a single target locus was integrated into the genome of 293 cells using retroviral transduction (Figure 3a). The extent of chromosomal gene targeting was determined seven days after co-transfection of each of the different ZFN expression vectors with the repair plasmid (Figure 3b). In all three target cell lines, only a few cells turned green in the absence of an expressed nuclease. Expression of I-SceI stimulated gene targeting on average by 50-fold, which is in good agreement with previously published results.9,10,35,36,13 After transfection of ZFNs harboring the EB0, HV0 ZF domains, the number of cells undergoing HR between the target locus and the repair plasmid remained low. While BA0-N, BA1-N, BA2-N and HV1-N revealed intermediate activity, cells transfected with ZFNs based on the EB1, EB2, and HV2 ZF domains stimulated HR to similar degrees as I-SceI. To evaluate ZFN performance further, the amount of transfected ZFN expression vectors was increased from 100 to 300 ng (Figure 3c). In the EB/EB and HV/HV target cell lines, gene targeting could only be marginally increased, if at all, when transfecting more of the nuclease expression plasmids. Augmenting the quantity of the BA1-N expression plasmid, however, doubled the number of EGFP-positive cells, closing in on the activity of the other nucleases. Although ZFN performance in the HR assays does not correlate completely with the in vitro DNA-binding parameters, the data suggests that both high affinity and high specificity are required for efficient ZFN activity in human cells. For example, stimulation of gene targeting by BA1-N and BA2-N was higher than that by BA0-N, although the BA0 domain possesses a higher DNA-binding affinity than BA1 and BA2. To verify that EGFP expression in gene corrected cells was a result of a genuine HR event, we performed genotype analysis. Two sets of primers were designed in such a way that only sequences contained in the corrected lacZ-EGFP target locus could be amplified (Figure 3a). As an example, a nested polymerase chain reaction (PCR) was performed on genomic DNA isolated from non-selected 293EB/EB target cells after transfection with expression vectors for www.moleculartherapy.org vol. 16 no. 2 feb. 2008
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0.6 0.4
**
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EB2
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Scel
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cto
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–2.0 –
+
+
+
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+
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–
RP
Figure 3 Zinc-finger nuclease (ZFN)-based stimulation of homologous recombination (HR) on chromosomal target loci. (a) Schematic. The illustration shows the design of the repair plasmid (RP) and the cellular target locus (TL), which was introduced by retroviral transduction. The corrected target locus (corr) is a product of legitimate HR between the TL and a RP. LTR, long-terminal repeat promoter; IRES, internal ribosomal entry site; wpre, woodchuck hepatitis virus post-transcription regulatory element. (b) Chromosomal gene targeting. 293 cells harboring a stably integrated TL were transfected with 100 ng of the nuclease expression plasmids, RV, and a REx expression vector to normalize for transfection efficiency. The columns designate the fraction of enhanced green fluorescent protein (EGFP)-positive cells 7 days post-transfection relative to I-SceI-mediated gene targeting. “cto” indicates transfection with a control vector. (c) Titration. The 293-based target cells were transfected as above but with two amounts of the ZFN expression plasmids (100, 300 ng). For 293HV/HV and 293BA/BA target cells, I-SceI-mediated gene targeting was only determined with 100 ng plasmid. The columns display the percentage of EGFP-positive cells in relation to transfected cells. For b and c, a statistically significant increase of HR as compared to non-stimulated HR (cto) is indicated by * (P < 0.03) and ** (P < 0.005). (d) Genotyping. Genomic DNA was isolated from 293EB/EB target cells after transfection with RP and the nuclease expression vectors. A nested polymerase chain reaction (PCR) was performed with primers designed to amplify a 2.1-kb product only from the corrected target locus. The positions of the primers used for the nested PCR protocol are indicated in a. Non-transfected target cells and the parental 293 cells served as controls.
I-SceI or the EB-based ZFNs (Figure 3d). No PCR products were detected from samples transfected without nuclease (cto) or with Molecular Therapy vol. 16 no. 2 feb. 2008
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b 180
**
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wpre LTR
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b
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Relative cell survival
a
Specificity, Activity, and Toxicity of ZFN
Figure 4 Zinc-finger nuclease (ZFN)-associated toxicity. (a) Cell survival assay. 293T cells were co-transfected with pEGFP and ZFN expression vectors as indicated. The percentage of enhanced green fluorescent protein (EGFP) positive cells was determined by flow cytometry 30 hours and 5 days later. The columns represent the fraction of positive cells at day 5 as compared to 30 hours post-transfection and is shown relative to transfection with a control (cto) plasmid encoding a non-functional nuclease. A statistically significant increase in survival as compared to EB0-N or BA0-N, respectively, is indicated by * (P < 0.02) or ** (P < 0.005). (b) Intracellular γ-H2AX levels. HT-1080 cells were transfected with ZFN expression vectors and cellular γ-H2AX levels were determined after 30 hours by flow cytometry using a γ-H2AX-specific antibody. The histogram shows a representative experiment involving BA-based ZFNs. (c) Genotoxicity assay. After flow cytometric analysis of cellular γ-H2AX levels, a gate was set arbitrarily to encompass ~1% of cells transfected with a control vector. The columns denote the number of highly γ-H2AX-positive cells as fraction of transfected cells. Statistically significant reduction in γ-H2AX levels as compared to EB0-N or BA0-N, respectively, is indicated by *** (P < 0.02).
EB0-N, whereas a product was amplified from cells following expression of I-SceI, EB1-N and EB2-N. These data confirm the results of the recombination assays and demonstrate that EGFP-positive cells arise as a result of legitimate HR between the target locus and a repair plasmid upon stimulation with a site-specific ZFN.
DNA-binding specificity inversely correlates with ZFN-induced toxicity ZFN-induced cytotoxicity has been hypothesized to result from unintended off-target activity of the nucleases. To assess ZFNassociated cytotoxicity, 293T cells were co-transfected with ZFN 355
Specificity, Activity, and Toxicity of ZFN
expression plasmids and an EGFP marker plasmid. The fate of transfected cells was followed over time by determining the percentage of EGFP-positive cells 30 hours and 5 days post-transfection. Survival of transfected cells was calculated as the fraction of EGFPpositive cells at day 5 in relation to cells transfected with a control plasmid (Figure 4a). While overexpression of EB0-N reduced cell survival to 20% as compared to control or I-SceI expression, overexpression of EB1-N or EB2-N showed significantly increased cell survival. Similarly, BA1-N and BA2-N revealed significantly reduced toxicity as compared to BA0-N. Surprisingly, HV0-N seemed less toxic than HV1-N or HV2-N. To validate the toxicity data, a recently described assay to measure immediate genotoxicity was performed in the diploid human cell line HT-1080.19 This assay permits quantification of the relative number of DSBs in cells expressing a ZFN of interest by measuring the level of γ-H2AX, a major component of DSB-induced repair foci.37 An original histogram record portraying the intracellular γ-H2AX levels is shown for the BA-based ZFNs (Figure 4b). Overexpression of EB1-N or EB2-N induced a significantly reduced level of γ-H2AX as compared to EB0-N (Figure 4c), suggesting that EB1-N and EB2-N created less offtarget DSBs than EB0-N. Similarly BA1-N and BA2-N induced lower levels of γ-H2AX than did BA0-N. Again, HV0-N was less toxic than HV1-N or HV2-N. The two data sets are in good accordance and imply an inverse relationship between the specificity of DNA-binding and ZFNassociated toxicity, when comparing proteins that bind to the same target DNA sequence. Of the three ZFNs that bind to the EB target site, EB1-N and EB2-N, which harbor ZF domains with a higher DNA-binding specificity, exhibit a lower toxicity. A similar trend is seen with the three ZFNs engineered to bind the BA target site. Interestingly, the HV0-N protein showed little toxicity relative to HV1-N and HV2-N (both of which possess higher affinities and specificities) in both assays. We hypothesize that this relatively lower toxicity stems from HV0’s lack of DNA-binding activity, as measured in vitro (see Figure 1a).
Discussion The ability to purposefully modify the genome of a living cell not only facilitates the study of gene function but also opens the door to targeted therapeutic interventions in the genomes of stem cells from patients suffering from inherited disorders. This report demonstrates for the first time that DNA-binding specificity, in addition to affinity, is a major determinant of ZFN performance. For a given target DNA site, ZFNs that induce the highest stimulation of gene targeting are those that harbor a ZF domain with both high affinity and high specificity. Importantly, this study also identifies DNA-binding specificity as an important determinant of ZFN-induced toxicity. We note, however, that the absolute in vitro DNA-binding specificity is not the only determinant of genotoxicity in human cells. The high toxicity observed for HV1-N, whose DNA-binding domain revealed the highest in vitro affinity and specificity among the nine ZF proteins we tested, suggests that additional factors may be critical. We speculate that the relative abundance and location of exactmatch or partial-match target sites (e.g., in vital genes) in the cellular genome has a decisive impact on ZFN performance and 356
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toxicity but further experiments are necessary to resolve these additional contributions. An important question when generating ZFNs is how one can rapidly evaluate the engineered ZF domains for their efficacy in human cells. Ideally, a rapid screening assay would identify ZF domains likely to exhibit high activity and minimal toxicity when expressed as ZFNs for any given target site of interest. Determination of in vitro affinities is laborious and not suitable for routine use. To address this issue, we tested different assays, which were previously suggested to be rapid screening methods for assessing the quality of ZF domains: a human cell-based transcriptional activator assay,13 a B2H assay,33 and an episomal recombination assay.13 In all of these assays, binding of the ZF domain to its DNA target site triggers a biological response that can be quantified by measuring the expression of a reporter gene. Although all assays are valuable predictors for the quality of the ZF domains, the human cell-based transcriptional reporter assay did not always correctly identify ZF domains with the best specificity for a given target DNA site. On the contrary, in the B2H system, in which the target site was present on a single-copy mini F’ reporter plasmid,38 reporter gene activation corresponded well with the in vitro affinity and specificity data. Moreover, for each of the three sites we tested, activity in the B2H assay generally correlated well with the activity, but not necessarily toxicity (see above), of the corresponding ZFNs in human cells, which establishes this assay as a good predictor of the fitness of a particular ZF domain to work as ZFN in human cells. Comparison of the results obtained with the simplified version of the B2H system used in this study38 with the previously published B2H data33 show that the two systems give virtually identical results, thereby validating the newer system. Notably—but not surprisingly—the episomal recombination assay most closely reproduced the results of ZFNstimulated chromosomal gene targeting. This report provides the first demonstration that ZF engineering strategies, which account for context-dependent DNAbinding effects, yield multi-finger domains that show high activity and low toxicity as ZFNs in human cells. In contrast to the modular assembly strategy,39 which treats ZFs as completely independent units, context-sensitive selection strategies40,41,33 account for potential context-dependent effects, including cooperativity of ZF binding and occasional recognition of a fourth base in the target sequence.20,42,21,43–45 Here we show that ZFNs based on ZF domains generated by a context-sensitive parallel optimization method (BA1, BA2, EB1, EB2, HV1, and HV2)33 generally reveal both good performance in stimulating gene targeting and low ZFN-induced genotoxicity. In the future, it will be important to perform largescale surveys assessing the “success rates” of context-sensitive parallel optimization and other ZF engineering methods, with the goal of providing researchers with the information that will allow them to make informed choices about which strategies are best suited for their needs. In this study we have defined a platform of assays that can be used to characterize ZF-based DNA-binding domains and ZFNs for their suitability to be employed in human cells. The simplified B2H assay has proven to reliably identify DNA-binding domains with high affinities and specificities for a target site of interest and the plasmid-based recombination assays permit fast www.moleculartherapy.org vol. 16 no. 2 feb. 2008
© The American Society of Gene Therapy
quantification of ZFN performance. Nonetheless, because factors in addition to the DNA-binding parameters can determine ZFN-associated toxicity, a toxicity assay remains an important component of the characterization process. Lastly, we note that ZFs that score well in our screening assays can also be fused to asymmetric, heterodimeric FokI cleavage domains19 to improve further the specificity of a pair of ZFNs. This combination of increasing both specificity of DNA binding and DNA cleavage should allow the generation of ZFNs that are well suited for applications in preclinical or clinical studies.
Materials and Methods Plasmids. Most plasmids were assembled by PCR and oligonucleotide-
directed cloning. Relevant sequences are given in Figures 1a and 2c. The B2H vectors expressing the various ZF domains fused to a fragment of the yeast Gal11P protein and pAC-Kan-alphaGal4 have been previously described.33,38 Single-copy B2H reporter plasmids were constructed by inserting pairs of oligonucleotides bearing the BCR-ABL, erbB2, or human immunodeficiency virus target sites into plasmid pBAC-lacZ.38 The resulting plasmids were named pBAC-X-lacZ, where X is the target site. Mammalian expression vectors for the ZF transcription factors were generated by subcloning the respective PCR amplified ZF domains EBx, HVx, and BAx (x standing for 0, 1, or 2) into vector pRK5.AD.46 Mammalian reporter plasmids were constructed by inserting a pair of annealed oligonucleotides bearing the combined HV/BA or two EB target sites into ptk.Luc47 to generate plasmids pxx.tk.Luc (xx refers to the binding site). Repair vector pUC. Zgfp/Rex and expression vectors pRK5.LHA-Sce1 and pCMV.Luc have been described previously.13 Target plasmids pCMV.LacZsXX∂GFP (XX denoting EB, BA, or HV) were generated by inserting the respective binding sites into plasmid pCMV.LacZs∂GFP.13 The neighboring nucleotides of the target sequences are shown in Figure 2c. Repair vector pUC.ZgfpINwpre was constructed by subcloning an IRES-NeoR-wpre cassette downstream of the EGFP open reading frame in pUC.Zgfp.13 The ZFN expression vectors are based on pRK5.GZF3-N and pPGK.GZF3-N13 and were generated by exchanging GZF3 with the respective PCR amplified ZF domains EBx, HVx, and BAx. Retroviral vector plasmids pS11.LacZsXX∂GFPiNwpre were obtained by subcloning the corresponding LacZXX∂GFP cassettes into pS11.EGiN (gift of Helmut Hanenberg, Düsseldorf). Maps and sequences of plasmids are available upon request. Transcriptional activation assays. The B2H transcriptional activation
assays were performed by transforming bacterial strain KJBAC138 with a B2H reporter plasmid (pBAC-X-lacZ). The resulting reporter strain cells were then doubly transformed with pAC-Kan-alphaGal4 and a B2H vector expressing the test ZF domain of interest fused to a Gal11P fragment. Transformants were assayed for β-galactosidase as previously described.48 Fold-activation values were calculated by comparing β-galactosidase values from cells expressing a ZF domain-Gal11P fusion to cells expressing only a Gal11P fragment. Human cell-based reporter assays were basically performed as previously described.13 Briefly, 293 cells in 12-well plates were transfected using calcium phosphate with a total of 2 µg of DNA which included: 0.25 µg of plasmid pRK5.ADXXx encoding a ZF transcription factor, 0.25 µg of the corresponding reporter plasmid pxx.tk.Luc, 0.0025 µg of pRL-CMV (expressing Renilla luciferase to normalize for transfection efficiency), 0.4975 µg of pCMV-β, and 1 µg of pUC118. Cells were harvested 40 hours after transfection in passive lysis buffer (Promega, Madison, WI) and firefly and Renilla luciferase activities were measured in a luminometer using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI). Fold-activation was calculated by comparing normalized firefly luciferase values from cells expressing a ZF-activator with cells expressing an empty vector.
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Retroviral vectors and generation of target cell lines. Retroviral vectors
to establish the three target cell lines were obtained by transfecting Phoenix cells in a 10 cm dish with 3 µg of pMD-G (gift of Inder Verma, La Jolla), 5 µg pM57 (gift of Christopher Baum, Hannover) and 10 µg of pS11.LacZsXX∂GFPiNwpre (XX denoting EB, BA, or HV) by the calcium phosphate method. Viral supernatants were collected 48 hours later, passed through a 45-µm filter to remove producer cells, and used to transduce 293 cells. The presence of a single-copy target locus was ensured by transducing cells with a predefined vector dose, which rendered less than 1% of cells LacZ-positive, as determined by X-Gal staining. Individual cells were clonally expanded in the presence of 0.4 mg/ml Geneticin (Gibco, Invitrogen, Karlsruhe, Germany). All cell lines were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum. Cell-based recombination assays and immunoblotting. The plasmid-
based recombination assays and immunoblotting were performed as previously described.13,19 For the chromosomal HR assay, 293-based target cells in 12-well plates were transfected by calcium phosphate precipitation with 2 µg of the repair plasmid (pUC.ZgfpINwpre), 100 or 300 ng of a cytomegalovirus (CMV)-driven nuclease expression vector or control vector (pCMV. Luc), and 10 ng of pDsRed-Express-N1 (Clontech, Mountain view, CA). Three and seven days after transfection, 50,000 cells were analyzed by flow cytometry to determine the percentage of EGFP- and REx-positive cells. The number of REx-positive cells at day 3 was used to normalize for transfection efficiency. Quantitative toxicity assay. For cell survival assay, 293T cells in 12-well
plates were transfected with 400 ng of a CMV-controlled nuclease expression vector, 100 ng of pEGFP (Clontech, Mountain view, CA), and pUC118 to 2 µg using calcium phosphate precipitation. The fraction of EGFP-positive cells was determined by flow cytometry after 30 hours and 5 days. The genotoxicity assay in HT-1080 cells was performed as previously described.19 Statistical analysis. All experiments were performed at least three times.
Statistical significance was determined using a one-sided (Figure 4) or two-sided (Figures 1–3) student’s t-test with unequal variance. Error bars represent SD.
Acknowledgments We thank Christopher Baum, Helmut Hanenberg, and Inder Verma for plasmids. This work was supported by grants CA311/1 and CA311/2 from the German Research Foundation, DFG (T.C.), R01 grant GM069906 from the National Institutes of Health (J.K.J.), the MGH Department of Pathology (J.K.J.) and fellowships from the Swiss National Science Foundation (T.I.C.) and the Fürst-Dietrichstein’sche-Stiftung (M.E.). Requests for B2H system reagents should be addressed to J.K.J. (jjoung@ partners.org); all other reagent requests should be addressed to T.C.
References
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9. Choulika, A, Perrin, A, Dujon, B and Nicolas, JF (1995). Induction of homologous recombination in mammalian chromosomes by using the I-SceI system of Saccharomyces cerevisiae. Mol Cell Biol 15: 1968–1973. 10. Smih, F, Rouet, P, Romanienko, PJ and Jasin, M (1995). Double-strand breaks at the target locus stimulate gene targeting in embryonic stem cells. Nucleic Acids Res 23: 5012–5019. 11. Bibikova, M, Beumer, K, Trautman, JK and Carroll, D (2003). Enhancing gene targeting with designed zinc finger nucleases. Science 300: 764. 12. Porteus, MH and Baltimore, D (2003). Chimeric nucleases stimulate gene targeting in human cells. Science 300: 763. 13. Alwin, S, Gere, MB, Guhl, E, Effertz, K, Barbas, CF 3rd, Segal, DJ et al. (2005). Custom zinc-finger nucleases for use in human cells. Mol Ther 12: 610–617. 14. Urnov, FD, Miller, JC, Lee, YL, Beausejour, CM, Rock, JM, Augustus, S et al. (2005). Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature 435: 646–651. 15. Wright, DA, Townsend, JA, Winfrey, RJ Jr., Irwin, PA, Rajagopal, J, Lonosky, PM et al. (2005). High-frequency homologous recombination in plants mediated by zinc-finger nucleases. Plant J 44: 693–705. 16. Beumer, K, Bhattacharyya, G, Bibikova, M, Trautman, JK and Carroll, D (2006). Efficient gene targeting in Drosophila with zinc-finger nucleases. Genetics 172: 2391–2403. 17. Porteus, MH (2006). Mammalian gene targeting with designed zinc finger nucleases. Mol Ther 13: 438–446. 18. Miller, JC, Holmes, MC, Wang, J, Guschin, DY, Lee, YL, Rupniewski, I et al. (2007). An improved zinc-finger nuclease architecture for highly specific genome editing. Nat Biotechnol 25: 778–785. 19. Szczepek, M, Brondani, V, Buchel, J, Serrano, L, Segal, DJ and Cathomen, T (2007). Structure-based redesign of the dimerization interface reduces the toxicity of zinc-finger nucleases. Nat Biotechnol 25: 786–793. 20. Pavletich, NP and Pabo, CO (1991). Zinc finger-DNA recognition: crystal structure of a Zif268-DNA complex at 2.1 A. Science 252: 809–817. 21. Elrod-Erickson, M, Rould, MA, Nekludova, L and Pabo, CO (1996). Zif268 protein-DNA complex refined at 1.6 A: a model system for understanding zinc finger-DNA interactions. Structure 4: 1171–1180. 22. Choo, Y and Klug, A (1994). Selection of DNA binding sites for zinc fingers using rationally randomized DNA reveals coded interactions. Proc Natl Acad Sci USA 91: 11168–11172. 23. Rebar, EJ and Pabo, CO (1994). Zinc finger phage: affinity selection of fingers with new DNA-binding specificities. Science 263: 671–673. 24. Wu, H, Yang, WP and Barbas, CF 3rd (1995). Building zinc fingers by selection: toward a therapeutic application. Proc Natl Acad Sci USA 92: 344–348. 25. Jamieson, AC, Wang, H and Kim, SH (1996). A zinc finger directory for high-affinity DNA recognition. Proc Natl Acad Sci USA 93: 12834–12839. 26. Segal, DJ, Dreier, B, Beerli, RR and Barbas, CF 3rd (1999). Toward controlling gene expression at will: selection and design of zinc finger domains recognizing each of the 5′-GNN-3′ DNA target sequences. Proc Natl Acad Sci USA 96: 2758–2763. 27. Dreier, B, Beerli, RR, Segal, DJ, Flippin, JD and Barbas, CF, 3rd (2001). Development of zinc finger domains for recognition of the 5′-ANN-3′ family of DNA sequences and their use in the construction of artificial transcription factors. J Biol Chem 276: 29466–29478. 28. Liu, Q, Xia, Z, Zhong, X and Case, CC (2002). Validated zinc finger protein designs for all 16 GNN DNA triplet targets. J Biol Chem 277: 3850–3856. 29. Blancafort, P, Magnenat, L and Barbas, CF 3rd (2003). Scanning the human genome with combinatorial transcription factor libraries. Nat Biotechnol 21: 269–274.
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30. Dreier, B, Fuller, RP, Segal, DJ, Lund, CV, Blancafort, P, Huber, A et al. (2005). Development of zinc finger domains for recognition of the 5′-CNN-3′ family DNA sequences and their use in the construction of artificial transcription factors. J Biol Chem 280: 35588–35597. 31. Bitinaite, J, Wah, DA, Aggarwal, AK and Schildkraut, I (1998). FokI dimerization is required for DNA cleavage. Proc Natl Acad Sci USA 95: 10570–10575. 32. Bibikova, M, Carroll, D, Segal, DJ, Trautman, JK, Smith, J, Kim, YG et al. (2001). Stimulation of homologous recombination through targeted cleavage by chimeric nucleases. Mol Cell Biol 21: 289–297. 33. Hurt, JA, Thibodeau, SA, Hirsh, AS, Pabo, CO and Joung, JK (2003). Highly specific zinc finger proteins obtained by directed domain shuffling and cell-based selection. Proc Natl Acad Sci USA 100: 12271–12276. 34. Joung, JK, Ramm, EI and Pabo, CO (2000). A bacterial two-hybrid selection system for studying protein-DNA and protein–protein interactions. Proc Natl Acad Sci USA 97: 7382–7387. 35. Miller, DG, Petek, LM and Russell, DW (2003). Human gene targeting by adeno-associated virus vectors is enhanced by DNA double-strand breaks. Mol Cell Biol 23: 3550–3557. 36. Porteus, MH, Cathomen, T, Weitzman, MD and Baltimore, D (2003). Efficient gene targeting mediated by adeno-associated virus and DNA double-strand breaks. Mol Cell Biol 23: 3558–3565. 37. Rogakou, EP, Pilch, DR, Orr, AH, Ivanova, VS and Bonner, WM (1998). DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139. J Biol Chem 273: 5858–5868. 38. Wright, DA, Thibodeau-Beganny, S, Sander, JD, Winfrey, RJ, Hirsh, AS, Eichtinger, M et al. (2006). Standardized reagents and protocols for engineering zinc finger nucleases by modular assembly. Nat Protocols 1: 1637. 39. Segal, DJ, Beerli, RR, Blancafort, P, Dreier, B, Effertz, K, Huber, A et al. (2003). Evaluation of a modular strategy for the construction of novel polydactyl zinc finger DNA-binding proteins. Biochemistry 42: 2137–2148. 40. Greisman, HA and Pabo, CO (1997). A general strategy for selecting high-affinity zinc finger proteins for diverse DNA target sites. Science 275: 657–661. 41. Isalan, M, Klug, A and Choo, Y (2001). A rapid, generally applicable method to engineer zinc fingers illustrated by targeting the HIV-1 promoter. Nat Biotechnol 19: 656–660. 42. Desjarlais, JR and Berg, JM (1993). Use of a zinc-finger consensus sequence framework and specificity rules to design specific DNA binding proteins. Proc Natl Acad Sci USA 90: 2256–2260. 43. Kim, CA and Berg, JM (1996). A 2.2 A resolution crystal structure of a designed zinc finger protein bound to DNA. Nat Struct Biol 3: 940–945. 44. Isalan, M, Klug, A and Choo, Y (1998). Comprehensive DNA recognition through concerted interactions from adjacent zinc fingers. Biochemistry 37: 12026–12033. 45. Dreier, B, Segal, DJ and Barbas, CF 3rd (2000). Insights into the molecular recognition of the 5′-GNN-3′ family of DNA sequences by zinc finger domains. J Mol Biol 303: 489–502. 46. Cathomen, T, Stracker, TH, Gilbert, LB and Weitzman, MD (2001). A genetic screen identifies a cellular regulator of adeno-associated virus. Proc Natl Acad Sci USA 98: 14991–14996. 47. Cathomen, T, Collete, D and Weitzman, MD (2000). A chimeric protein containing the N terminus of the adeno-associated virus Rep protein recognizes its target site in an in vivo assay. J Virol 74: 2372–2382. 48. Thibodeau, SA, Fang, R and Joung, JK (2004). High-throughput β-galactosidase assay for bacterial cell-based reporter systems. Biotechniques 36: 410–415.
www.moleculartherapy.org vol. 16 no. 2 feb. 2008
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DNA-binding Specificity Is a Major Determinant of the Activity and Toxicity of Zinc-finger Nucleases Tatjana I Cornu1, Stacey Thibodeau-Beganny2, Eva Guhl1, Stephen Alwin1, Magdalena Eichtinger2,3, JK Joung2,3 and Toni Cathomen1 1 Charité Medical School, Institute of Virology (CBF), Berlin, Germany; 2Molecular Pathology Unit, Center for Cancer Research, and Center for omputational and Integrative Biology, Massachusetts General Hospital, Charlestown, Massachusetts, USA; 3Department of Pathology, C Harvard Medical School, Boston, Massachusetts, USA
The engineering of proteins to manipulate cellular genomes has developed into a promising technology for biomedical research, including gene therapy. In particular, zinc-finger nucleases (ZFNs), which consist of a nonspecific endonuclease domain tethered to a tailored zinc-finger (ZF) DNA-binding domain, have proven invaluable for stimulating homology-directed gene repair in a variety of cell types. However, previous studies demonstrated that ZFNs could be associated with significant cytotoxicity due to cleavage at off-target sites. Here, we compared the in vitro affinities and specificities of nine ZF DNA-binding domains with their performance as ZFNs in human cells. The results of our cell-based assays reveal that the DNA-binding specificity—in addition to the affinity—is a major determinant of ZFN activity and is inversely correlated with ZFN-associated toxicity. In addition, our data provide the first evidence that engineering strategies, which account for context-dependent DNAbinding effects, yield ZFs that function as highly efficient ZFNs in human cells. Received 1 September 2007; accepted 15 October 2007; published online 20 November 2007. doi:10.1038/sj.mt.6300357
Introduction Engineered proteins that permit the targeted modulation of a cellular genome represent an important tool for genetic studies, biotechnology, and gene therapy.1–5 Zinc-finger nucleases (ZFNs) are customizable artificial nucleases composed of an engineered zinc-finger (ZF) domain linked to the cleavage domain of the type IIs restriction endonuclease FokI.6,7 ZFNs can be engineered to bind and cleave chromosomes at a desired locus and are therefore invaluable for stimulating site-specific genetic modifications based on homologous recombination (HR). HR is a rare event in mammalian cells,8 but the frequency can be dramatically improved by the introduction of a targeted DNA double-strand break (DSB) near the site of the desired recombination event.9,10 Several studies have reported gene targeting frequencies between 1 and 20% when a DSB was introduced by engineered ZFNs.11–19
To generate a ZFN directed against a specific DNA target sequence, a DNA-binding domain has to be engineered that contains three or four artificial ZF domains. Such ZFs are small domains of ~30 residues, which consist of a ββα-fold coordinated by a zinc ion. The structure of the transcription factor Zif268, which contains three ZF domains, revealed that each finger uses residues located at the amino-terminal end of its α-helix to specifically bind three to four bases of DNA.20,21 A large number of studies have shown that alteration of six amino acids in the αhelix is sufficient to create ZFs with novel DNA-binding specificities. Using a combination of rational design and/or selection strategies, a library of ZF modules has been created that includes DNA-binding domains able to recognize most of the 64 possible triplets.22–30 Individual ZFs can be assembled into tandem arrays, which can recognize a wide variety of novel target sites. Because ZFNs cleave DNA as dimers, two ZFN subunits are typically designed to recognize the target sequence in a tail-to-tail conformation.31,7 The C-terminal FokI domains are positioned in close proximity on the same face of the DNA helix by separating the two ZFN binding sites by a 5- or 6-base pair (bp) spacer.32 Previously published studies have reported that not all engineered ZF domains exhibit optimal behavior as ZFNs. While some of these ZFNs lack activity at all others induce significant cytotoxicity in human cells, an effect proposed to be caused by cleavage at “off-target” sites.12,13,17–19 ZFN-induced toxicity is likely determined by multiple factors, including the specificity of DNA binding and DNA cleavage as well as the frequency and location of sites in the genome actually bound by the ZFNs. We and others recently described a structure-based approach to reducing ZFN-induced toxicity.18,19 Rational redesign of the FokI domain was used to create an asymmetric dimer interface that destabilized dimerization and forced heterodimerization of the ZFN subunits.19 The resulting ZFNs elicited significantly reduced cytotoxicity without compromising on performance, suggesting that architectural changes in the ZFN structure can improve the specificity of DNA cleavage.18,19 Another study presented data suggesting that cytotoxicity could be reduced by identifying ZFN variants with “improved in vitro DNA-binding and cleavage properties”.14 However, the authors of this report did not specify the nature of the optimization performed.
Correspondence: Toni Cathomen, Charité Medical School, Institute of Virology (CBF), Hindenburgdamm 27, D-12203 Berlin, Germany. E-mail: [email protected]
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In this study, we sought to understand the relationship between the DNA-binding affinities and specificities of ZF domains and their performance and toxicity as ZFNs in human cells. To this end, we examined various ZFNs engineered to bind to the same sites but which possess different DNA-binding affinities and specificities. Our results demonstrate that for a given DNA target site, both the DNA-binding affinity and specificity of the ZF domain influence the activity and toxicity of a ZFN. Our data also provide the first evidence that ZF domains engineered using a bacterialtwo-hybrid (B2H)-based selection strategy function well as ZFNs in human cells.
Results Evaluation of engineered ZF domains using transcriptional activation assays To assess the relationship between the in vitro DNA-binding affinity and specificity of engineered ZF domains and their performance as ZFNs in human cells, we chose a series of nine
a ZF
Target site
Zinc-finger sequences (–1 to +6)
KdS mol/l
KdN nmol/l
Specificity
EB0 5′EB1 EB2
150 31 65
1,000 1,100 1,100
6,700 35,000 17,000
HV0 5′HV1 HV2
– 9 9
– 1,100 180
– 87,000 19,000
BA0 5′BA1 BA2
28 78 60
55 2,100 1,300
1,980 27,000 23,000
AD
b 3
2
1
Mammalia 30
EB/EB
HV/BA
*
25
*
20
*
*
*
G11
c 3
2
BA0
cto
HV2
HV1
G4
cto
EB1
EB0
cto
HV0
**
5 0
*
BA2
10
BA1
15
EB2
Fold-activation
Luciferase
TATA
1
R�
Bacteria
–35/–10
lacZ
25
**
**
15 10
*
EB
*
HV
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BA
BA2
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cto
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HV0
** cto
EB2
cto
0
*
EB1
5
** **
EB0
Fold-activation
20
previously described ZF domains directed against target sites in the erbB2 locus (EB), a BCR-ABL translocation sequence (BA), or the human immunodeficiency virus-1 promoter (HV).33 For each of the three DNA target sequences three ZF domains were generated, each possessing varying affinities and specificities for their binding sites (Figure 1a). We initially characterized these nine ZF domains using two different transcriptional reporter assays designed to serve as surrogate assays of DNA binding in cells: one based in human cells and the other in bacteria. For the human cell-based assay, we constructed expression vectors encoding each of the nine different ZF domains fused to the VP16 transcriptional activation domain to create artificial transcription factors. Each of these ZF-activatorencoding plasmids were transiently transfected into human 293T cells together with a luciferase reporter bearing either a cognate or non-cognate upstream binding site for the ZF to be evaluated (Figure 1b). As expected based on the in vitro DNA-binding data, the EB1 and EB2 activators stimulated transcription from their specific reporter more efficiently than the EB0 activator, and the HV1 and HV2 activators performed better than the HV0 activator. Surprisingly, the BA0 activator stimulated luciferase expression more efficiently than either the BA1 or BA2 activators. These results suggest that within a set of ZF domains targeted to a given target site, transcriptional activation in the mammalian cellbased assay correlates well with the in vitro DNA-binding affinity (Figure 1a) of the ZF domain tested. To analyze the ZF domains in a B2H assay, the ZFs of interest were fused to a fragment of the yeast Gal11P protein and expressed in bacterial cells harboring a single-copy reporter vector. The reporter bears the cognate ZF target sequence upstream of a weak promoter, which directs expression of a lacZ reporter gene (Figure 1c). These reporter cells also express a second hybrid protein consisting of an RNA polymerase α-subunit domain fused Figure 1 Characteristics of engineered zinc-finger (ZF) domains. (a) In vitro data. The genomic DNA target sites and the sequences of the crucial residues in the recognition helix (positions −1 to +6) are indicated for each ZF module. The affinities and specificities have been determined previously.33 KdS, affinity to target site; KdN, affinity to nonspecific site; the specificity is the ratio of KdN/KdS. (b) Mammalian cellbased assay. For expression in human cells, the VP16 transcriptional activation domain (AD) is fused to the SV40 nuclear localization signal (black oval), and three ZF modules (1, 2, 3). The reporter plasmid contains the luciferase gene downstream of a minimal herpes simplex virus thymidine kinase promoter element (TATA), and upstream binding sites for the ZFs (grey boxes, each box represents 3 base pair of the target site). 293T cells were transfected with expression plasmids for the custom AD-ZF, reporter plasmids containing either two binding sites for the EB fingers (EB/EB) or a combined target site for the HV and BA fingers (HV/BA). Luciferase activity was normalized for transfection efficiency and is shown relative to transfection with empty vector (cto). (c) Bacterial cell-based assay. In the bacterial-two-hybrid (B2H) reporter system, expression of the lacZ reporter gene is dependent upon binding of the ZF–Gal11P (G11) hybrid protein to the target site. ZF–G11 interacts with the Gal4 (G4)-RNAP α-subunit (Rα) hybrid protein, which then recruits the RNA polymerase to the weak downstream promoter (−35/−10). The B2H reporter strains were doubly transformed with plasmids expressing the ZF–G11 and G4–Rα hybrid proteins and transformants were assayed for β-galactosidase activity. Fold-activation of lacZ expression is shown relative to transfection with empty vector (cto). Statistical significance for both assays is indicated by * (P < 0.007) and ** (P < 0.001).
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to a fragment of the yeast Gal4 protein, known to interact with Gal11P. In contrast to the results obtained with the mammalian system, the BA0 domain activated transcription less efficiently than the BA1 or BA2-based activators, suggesting that the DNAbinding specificity—in addition to affinity—is a determinant of the activity of a ZF domain in the B2H system.34,33
DNA-binding specificity contributes to ZFN activity in human cells ZFN expression plasmids were generated by fusing each of the nine ZF domains to the cleavage domain of the FokI endonuclease (Figure 2a). Western blot analysis confirmed that all ZFNs were expressed at comparable steady-state levels in human cells (Figure 2b). A recently described recombination assay13 was used to quantify the activities of these nine ZFNs in human cells by measuring their abilities to stimulate HR between a target plasmid and a repair plasmid (Figure 2c). The target plasmid harbors a lacZ gene followed by stop codons and a 5′-truncated
a
HV2
HV1
HV0
BA2
BA1
BA0
EB2
Fok l EB1
3 EB0
cto
b
2 I-Scel
1
33 ZFN 25
c ∂LacZ
EGFP ∂GFP
LacZ
CMV
SV40 SV40
REx
RP
Neo
TP
I-Scel
EB EB
*
*
**
50 40
*
30 20
BA2
BA1
BA0
Scel
cto
HV2
HV1
HV0
Scel
cto
EB2
EB1
cto
EB0
*
10 0
*
*
Scel
% GFP-positive cells
d 60
Figure 2 Stimulation of episomal gene targeting by designer zincfinger nucleases (ZFNs). (a) Design of ZFNs. All ZFNs contain an Nterminal HA tag (small oval), followed by three ZF modules (1, 2, 3), a short AAARA linker and the catalytic domain of FokI. (b) ZFN expression levels. Transfected 293T cells were harvested after 30 hours and lysates probed with an antibody against the HA tag. “cto” indicates transfection with a control vector. Size markers are indicated on the left. (c) Experimental set up. Repair plasmid (RP) and target plasmid (TP) are described in the text. The recognition site for I-SceI and the homodimeric binding sites for the EB-based ZFNs are highlighted. (d) Episomal gene targeting. 293T cells were transfected with RP, TP and nuclease expression vectors. The columns designate the percentage of enhanced green fluorescent protein (EGFP)-positive cells 2 days post-transfection as determined by flow cytometry. CMV, cytomegalovirus. Promoter; SV40, Simian virus 40 promoter.
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enhanced green fluorescent protein (EGFP) gene (∂GFP), which ensures that no functional EGFP is expressed. The target sites for each of the three sets of ZFNs (EB, HV, BA) were placed as inverted repeats of the respective 9-bp binding sites between the lacZ and truncated EGFP open reading frames together with the cleavage site for the meganuclease I-SceI (as a control). The repair plasmid harbors a non-transcribed 5′-truncated lacZEGFP fusion gene and is designed to rescue EGFP expression through HR by generating a lacZ-EGFP fusion gene. For all target loci, only a few cells turned green in the absence of an expressed nuclease (cto), representing non-stimulated gene targeting (Figure 2d). Expression of EB0-based ZFN, EBO, stimulated HR only poorly, whereas expression of EB1-N and EB2-N stimulated gene correction to a similar extent as I-SceI, with some 40% of transfected cells expressing a functional lacZ-EGFP gene. (Note that 40% of EGFP-positive cells does not correspond to conversion of 40% of the target DNAs because each cell contains many plasmids.) Similarly, expression of HV1-N and HV2N as well as BA1-N and BA2-N stimulated HR to a larger extent than HV0-N and BA0-N, respectively. We then assessed performance of the nine different ZFNs using a chromosomal version of our HR assay. To do this, we generated specifically designed target cell lines, in which a single target locus was integrated into the genome of 293 cells using retroviral transduction (Figure 3a). The extent of chromosomal gene targeting was determined seven days after co-transfection of each of the different ZFN expression vectors with the repair plasmid (Figure 3b). In all three target cell lines, only a few cells turned green in the absence of an expressed nuclease. Expression of I-SceI stimulated gene targeting on average by 50-fold, which is in good agreement with previously published results.9,10,35,36,13 After transfection of ZFNs harboring the EB0, HV0 ZF domains, the number of cells undergoing HR between the target locus and the repair plasmid remained low. While BA0-N, BA1-N, BA2-N and HV1-N revealed intermediate activity, cells transfected with ZFNs based on the EB1, EB2, and HV2 ZF domains stimulated HR to similar degrees as I-SceI. To evaluate ZFN performance further, the amount of transfected ZFN expression vectors was increased from 100 to 300 ng (Figure 3c). In the EB/EB and HV/HV target cell lines, gene targeting could only be marginally increased, if at all, when transfecting more of the nuclease expression plasmids. Augmenting the quantity of the BA1-N expression plasmid, however, doubled the number of EGFP-positive cells, closing in on the activity of the other nucleases. Although ZFN performance in the HR assays does not correlate completely with the in vitro DNA-binding parameters, the data suggests that both high affinity and high specificity are required for efficient ZFN activity in human cells. For example, stimulation of gene targeting by BA1-N and BA2-N was higher than that by BA0-N, although the BA0 domain possesses a higher DNA-binding affinity than BA1 and BA2. To verify that EGFP expression in gene corrected cells was a result of a genuine HR event, we performed genotype analysis. Two sets of primers were designed in such a way that only sequences contained in the corrected lacZ-EGFP target locus could be amplified (Figure 3a). As an example, a nested polymerase chain reaction (PCR) was performed on genomic DNA isolated from non-selected 293EB/EB target cells after transfection with expression vectors for www.moleculartherapy.org vol. 16 no. 2 feb. 2008
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Figure 3 Zinc-finger nuclease (ZFN)-based stimulation of homologous recombination (HR) on chromosomal target loci. (a) Schematic. The illustration shows the design of the repair plasmid (RP) and the cellular target locus (TL), which was introduced by retroviral transduction. The corrected target locus (corr) is a product of legitimate HR between the TL and a RP. LTR, long-terminal repeat promoter; IRES, internal ribosomal entry site; wpre, woodchuck hepatitis virus post-transcription regulatory element. (b) Chromosomal gene targeting. 293 cells harboring a stably integrated TL were transfected with 100 ng of the nuclease expression plasmids, RV, and a REx expression vector to normalize for transfection efficiency. The columns designate the fraction of enhanced green fluorescent protein (EGFP)-positive cells 7 days post-transfection relative to I-SceI-mediated gene targeting. “cto” indicates transfection with a control vector. (c) Titration. The 293-based target cells were transfected as above but with two amounts of the ZFN expression plasmids (100, 300 ng). For 293HV/HV and 293BA/BA target cells, I-SceI-mediated gene targeting was only determined with 100 ng plasmid. The columns display the percentage of EGFP-positive cells in relation to transfected cells. For b and c, a statistically significant increase of HR as compared to non-stimulated HR (cto) is indicated by * (P < 0.03) and ** (P < 0.005). (d) Genotyping. Genomic DNA was isolated from 293EB/EB target cells after transfection with RP and the nuclease expression vectors. A nested polymerase chain reaction (PCR) was performed with primers designed to amplify a 2.1-kb product only from the corrected target locus. The positions of the primers used for the nested PCR protocol are indicated in a. Non-transfected target cells and the parental 293 cells served as controls.
I-SceI or the EB-based ZFNs (Figure 3d). No PCR products were detected from samples transfected without nuclease (cto) or with Molecular Therapy vol. 16 no. 2 feb. 2008
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Specificity, Activity, and Toxicity of ZFN
Figure 4 Zinc-finger nuclease (ZFN)-associated toxicity. (a) Cell survival assay. 293T cells were co-transfected with pEGFP and ZFN expression vectors as indicated. The percentage of enhanced green fluorescent protein (EGFP) positive cells was determined by flow cytometry 30 hours and 5 days later. The columns represent the fraction of positive cells at day 5 as compared to 30 hours post-transfection and is shown relative to transfection with a control (cto) plasmid encoding a non-functional nuclease. A statistically significant increase in survival as compared to EB0-N or BA0-N, respectively, is indicated by * (P < 0.02) or ** (P < 0.005). (b) Intracellular γ-H2AX levels. HT-1080 cells were transfected with ZFN expression vectors and cellular γ-H2AX levels were determined after 30 hours by flow cytometry using a γ-H2AX-specific antibody. The histogram shows a representative experiment involving BA-based ZFNs. (c) Genotoxicity assay. After flow cytometric analysis of cellular γ-H2AX levels, a gate was set arbitrarily to encompass ~1% of cells transfected with a control vector. The columns denote the number of highly γ-H2AX-positive cells as fraction of transfected cells. Statistically significant reduction in γ-H2AX levels as compared to EB0-N or BA0-N, respectively, is indicated by *** (P < 0.02).
EB0-N, whereas a product was amplified from cells following expression of I-SceI, EB1-N and EB2-N. These data confirm the results of the recombination assays and demonstrate that EGFP-positive cells arise as a result of legitimate HR between the target locus and a repair plasmid upon stimulation with a site-specific ZFN.
DNA-binding specificity inversely correlates with ZFN-induced toxicity ZFN-induced cytotoxicity has been hypothesized to result from unintended off-target activity of the nucleases. To assess ZFNassociated cytotoxicity, 293T cells were co-transfected with ZFN 355
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expression plasmids and an EGFP marker plasmid. The fate of transfected cells was followed over time by determining the percentage of EGFP-positive cells 30 hours and 5 days post-transfection. Survival of transfected cells was calculated as the fraction of EGFPpositive cells at day 5 in relation to cells transfected with a control plasmid (Figure 4a). While overexpression of EB0-N reduced cell survival to 20% as compared to control or I-SceI expression, overexpression of EB1-N or EB2-N showed significantly increased cell survival. Similarly, BA1-N and BA2-N revealed significantly reduced toxicity as compared to BA0-N. Surprisingly, HV0-N seemed less toxic than HV1-N or HV2-N. To validate the toxicity data, a recently described assay to measure immediate genotoxicity was performed in the diploid human cell line HT-1080.19 This assay permits quantification of the relative number of DSBs in cells expressing a ZFN of interest by measuring the level of γ-H2AX, a major component of DSB-induced repair foci.37 An original histogram record portraying the intracellular γ-H2AX levels is shown for the BA-based ZFNs (Figure 4b). Overexpression of EB1-N or EB2-N induced a significantly reduced level of γ-H2AX as compared to EB0-N (Figure 4c), suggesting that EB1-N and EB2-N created less offtarget DSBs than EB0-N. Similarly BA1-N and BA2-N induced lower levels of γ-H2AX than did BA0-N. Again, HV0-N was less toxic than HV1-N or HV2-N. The two data sets are in good accordance and imply an inverse relationship between the specificity of DNA-binding and ZFNassociated toxicity, when comparing proteins that bind to the same target DNA sequence. Of the three ZFNs that bind to the EB target site, EB1-N and EB2-N, which harbor ZF domains with a higher DNA-binding specificity, exhibit a lower toxicity. A similar trend is seen with the three ZFNs engineered to bind the BA target site. Interestingly, the HV0-N protein showed little toxicity relative to HV1-N and HV2-N (both of which possess higher affinities and specificities) in both assays. We hypothesize that this relatively lower toxicity stems from HV0’s lack of DNA-binding activity, as measured in vitro (see Figure 1a).
Discussion The ability to purposefully modify the genome of a living cell not only facilitates the study of gene function but also opens the door to targeted therapeutic interventions in the genomes of stem cells from patients suffering from inherited disorders. This report demonstrates for the first time that DNA-binding specificity, in addition to affinity, is a major determinant of ZFN performance. For a given target DNA site, ZFNs that induce the highest stimulation of gene targeting are those that harbor a ZF domain with both high affinity and high specificity. Importantly, this study also identifies DNA-binding specificity as an important determinant of ZFN-induced toxicity. We note, however, that the absolute in vitro DNA-binding specificity is not the only determinant of genotoxicity in human cells. The high toxicity observed for HV1-N, whose DNA-binding domain revealed the highest in vitro affinity and specificity among the nine ZF proteins we tested, suggests that additional factors may be critical. We speculate that the relative abundance and location of exactmatch or partial-match target sites (e.g., in vital genes) in the cellular genome has a decisive impact on ZFN performance and 356
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toxicity but further experiments are necessary to resolve these additional contributions. An important question when generating ZFNs is how one can rapidly evaluate the engineered ZF domains for their efficacy in human cells. Ideally, a rapid screening assay would identify ZF domains likely to exhibit high activity and minimal toxicity when expressed as ZFNs for any given target site of interest. Determination of in vitro affinities is laborious and not suitable for routine use. To address this issue, we tested different assays, which were previously suggested to be rapid screening methods for assessing the quality of ZF domains: a human cell-based transcriptional activator assay,13 a B2H assay,33 and an episomal recombination assay.13 In all of these assays, binding of the ZF domain to its DNA target site triggers a biological response that can be quantified by measuring the expression of a reporter gene. Although all assays are valuable predictors for the quality of the ZF domains, the human cell-based transcriptional reporter assay did not always correctly identify ZF domains with the best specificity for a given target DNA site. On the contrary, in the B2H system, in which the target site was present on a single-copy mini F’ reporter plasmid,38 reporter gene activation corresponded well with the in vitro affinity and specificity data. Moreover, for each of the three sites we tested, activity in the B2H assay generally correlated well with the activity, but not necessarily toxicity (see above), of the corresponding ZFNs in human cells, which establishes this assay as a good predictor of the fitness of a particular ZF domain to work as ZFN in human cells. Comparison of the results obtained with the simplified version of the B2H system used in this study38 with the previously published B2H data33 show that the two systems give virtually identical results, thereby validating the newer system. Notably—but not surprisingly—the episomal recombination assay most closely reproduced the results of ZFNstimulated chromosomal gene targeting. This report provides the first demonstration that ZF engineering strategies, which account for context-dependent DNAbinding effects, yield multi-finger domains that show high activity and low toxicity as ZFNs in human cells. In contrast to the modular assembly strategy,39 which treats ZFs as completely independent units, context-sensitive selection strategies40,41,33 account for potential context-dependent effects, including cooperativity of ZF binding and occasional recognition of a fourth base in the target sequence.20,42,21,43–45 Here we show that ZFNs based on ZF domains generated by a context-sensitive parallel optimization method (BA1, BA2, EB1, EB2, HV1, and HV2)33 generally reveal both good performance in stimulating gene targeting and low ZFN-induced genotoxicity. In the future, it will be important to perform largescale surveys assessing the “success rates” of context-sensitive parallel optimization and other ZF engineering methods, with the goal of providing researchers with the information that will allow them to make informed choices about which strategies are best suited for their needs. In this study we have defined a platform of assays that can be used to characterize ZF-based DNA-binding domains and ZFNs for their suitability to be employed in human cells. The simplified B2H assay has proven to reliably identify DNA-binding domains with high affinities and specificities for a target site of interest and the plasmid-based recombination assays permit fast www.moleculartherapy.org vol. 16 no. 2 feb. 2008
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quantification of ZFN performance. Nonetheless, because factors in addition to the DNA-binding parameters can determine ZFN-associated toxicity, a toxicity assay remains an important component of the characterization process. Lastly, we note that ZFs that score well in our screening assays can also be fused to asymmetric, heterodimeric FokI cleavage domains19 to improve further the specificity of a pair of ZFNs. This combination of increasing both specificity of DNA binding and DNA cleavage should allow the generation of ZFNs that are well suited for applications in preclinical or clinical studies.
Materials and Methods Plasmids. Most plasmids were assembled by PCR and oligonucleotide-
directed cloning. Relevant sequences are given in Figures 1a and 2c. The B2H vectors expressing the various ZF domains fused to a fragment of the yeast Gal11P protein and pAC-Kan-alphaGal4 have been previously described.33,38 Single-copy B2H reporter plasmids were constructed by inserting pairs of oligonucleotides bearing the BCR-ABL, erbB2, or human immunodeficiency virus target sites into plasmid pBAC-lacZ.38 The resulting plasmids were named pBAC-X-lacZ, where X is the target site. Mammalian expression vectors for the ZF transcription factors were generated by subcloning the respective PCR amplified ZF domains EBx, HVx, and BAx (x standing for 0, 1, or 2) into vector pRK5.AD.46 Mammalian reporter plasmids were constructed by inserting a pair of annealed oligonucleotides bearing the combined HV/BA or two EB target sites into ptk.Luc47 to generate plasmids pxx.tk.Luc (xx refers to the binding site). Repair vector pUC. Zgfp/Rex and expression vectors pRK5.LHA-Sce1 and pCMV.Luc have been described previously.13 Target plasmids pCMV.LacZsXX∂GFP (XX denoting EB, BA, or HV) were generated by inserting the respective binding sites into plasmid pCMV.LacZs∂GFP.13 The neighboring nucleotides of the target sequences are shown in Figure 2c. Repair vector pUC.ZgfpINwpre was constructed by subcloning an IRES-NeoR-wpre cassette downstream of the EGFP open reading frame in pUC.Zgfp.13 The ZFN expression vectors are based on pRK5.GZF3-N and pPGK.GZF3-N13 and were generated by exchanging GZF3 with the respective PCR amplified ZF domains EBx, HVx, and BAx. Retroviral vector plasmids pS11.LacZsXX∂GFPiNwpre were obtained by subcloning the corresponding LacZXX∂GFP cassettes into pS11.EGiN (gift of Helmut Hanenberg, Düsseldorf). Maps and sequences of plasmids are available upon request. Transcriptional activation assays. The B2H transcriptional activation
assays were performed by transforming bacterial strain KJBAC138 with a B2H reporter plasmid (pBAC-X-lacZ). The resulting reporter strain cells were then doubly transformed with pAC-Kan-alphaGal4 and a B2H vector expressing the test ZF domain of interest fused to a Gal11P fragment. Transformants were assayed for β-galactosidase as previously described.48 Fold-activation values were calculated by comparing β-galactosidase values from cells expressing a ZF domain-Gal11P fusion to cells expressing only a Gal11P fragment. Human cell-based reporter assays were basically performed as previously described.13 Briefly, 293 cells in 12-well plates were transfected using calcium phosphate with a total of 2 µg of DNA which included: 0.25 µg of plasmid pRK5.ADXXx encoding a ZF transcription factor, 0.25 µg of the corresponding reporter plasmid pxx.tk.Luc, 0.0025 µg of pRL-CMV (expressing Renilla luciferase to normalize for transfection efficiency), 0.4975 µg of pCMV-β, and 1 µg of pUC118. Cells were harvested 40 hours after transfection in passive lysis buffer (Promega, Madison, WI) and firefly and Renilla luciferase activities were measured in a luminometer using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI). Fold-activation was calculated by comparing normalized firefly luciferase values from cells expressing a ZF-activator with cells expressing an empty vector.
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Retroviral vectors and generation of target cell lines. Retroviral vectors
to establish the three target cell lines were obtained by transfecting Phoenix cells in a 10 cm dish with 3 µg of pMD-G (gift of Inder Verma, La Jolla), 5 µg pM57 (gift of Christopher Baum, Hannover) and 10 µg of pS11.LacZsXX∂GFPiNwpre (XX denoting EB, BA, or HV) by the calcium phosphate method. Viral supernatants were collected 48 hours later, passed through a 45-µm filter to remove producer cells, and used to transduce 293 cells. The presence of a single-copy target locus was ensured by transducing cells with a predefined vector dose, which rendered less than 1% of cells LacZ-positive, as determined by X-Gal staining. Individual cells were clonally expanded in the presence of 0.4 mg/ml Geneticin (Gibco, Invitrogen, Karlsruhe, Germany). All cell lines were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum. Cell-based recombination assays and immunoblotting. The plasmid-
based recombination assays and immunoblotting were performed as previously described.13,19 For the chromosomal HR assay, 293-based target cells in 12-well plates were transfected by calcium phosphate precipitation with 2 µg of the repair plasmid (pUC.ZgfpINwpre), 100 or 300 ng of a cytomegalovirus (CMV)-driven nuclease expression vector or control vector (pCMV. Luc), and 10 ng of pDsRed-Express-N1 (Clontech, Mountain view, CA). Three and seven days after transfection, 50,000 cells were analyzed by flow cytometry to determine the percentage of EGFP- and REx-positive cells. The number of REx-positive cells at day 3 was used to normalize for transfection efficiency. Quantitative toxicity assay. For cell survival assay, 293T cells in 12-well
plates were transfected with 400 ng of a CMV-controlled nuclease expression vector, 100 ng of pEGFP (Clontech, Mountain view, CA), and pUC118 to 2 µg using calcium phosphate precipitation. The fraction of EGFP-positive cells was determined by flow cytometry after 30 hours and 5 days. The genotoxicity assay in HT-1080 cells was performed as previously described.19 Statistical analysis. All experiments were performed at least three times.
Statistical significance was determined using a one-sided (Figure 4) or two-sided (Figures 1–3) student’s t-test with unequal variance. Error bars represent SD.
Acknowledgments We thank Christopher Baum, Helmut Hanenberg, and Inder Verma for plasmids. This work was supported by grants CA311/1 and CA311/2 from the German Research Foundation, DFG (T.C.), R01 grant GM069906 from the National Institutes of Health (J.K.J.), the MGH Department of Pathology (J.K.J.) and fellowships from the Swiss National Science Foundation (T.I.C.) and the Fürst-Dietrichstein’sche-Stiftung (M.E.). Requests for B2H system reagents should be addressed to J.K.J. (jjoung@ partners.org); all other reagent requests should be addressed to T.C.
References
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