Zinc-finger Nuclease-induced Gene Repair With Oligodeoxynucleotides

original article

© The American Society of Gene & Cell Therapy

Zinc-finger Nuclease-induced Gene Repair With Oligodeoxynucleotides: Wanted and Unwanted Target Locus Modifications Sarah Radecke1, Frank Radecke2, Toni Cathomen3 and Klaus Schwarz1,2 Department of Molecular Diagnostics, Molecular Therapy and Experimental Transplantation, Institute for Clinical Transfusion Medicine and Immunogenetics Ulm, German Red Cross Blood Donation Service Baden-Wuerttemberg—Hessen, Ulm, Germany; 2Institute for Transfusion Medicine, University of Ulm, Ulm, Germany; 3Department of Experimental Hematology, Hannover Medical School, Hannover, Germany

1

Correcting a mutated gene directly at its endogenous locus represents an alternative to gene therapy protocols based on viral vectors with their risk of insertional mutagenesis. When solely a single-stranded oligodeoxynucleotide (ssODN) is used as a repair matrix, the efficiency of the targeted gene correction is low. However, as shown with the homing endonuclease I-SceI, ssODNmediated gene correction can be enhanced by concomitantly inducing a DNA double-strand break (DSB) close to the mutation. Because I-SceI is hardly adjustable to cut at any desired position in the human genome, here, customizable zinc-finger nucleases (ZFNs) were used to stimulate ssODN-mediated repair of a mutated single-copy reporter locus stably integrated into human embryonic kidney-293 cells. The ZFNs induced faithful gene repair at a frequency of 0.16%. Six times more often, ZFN-induced DSBs were found to be modified by unfaithful addition of ssODN between the termini and about 60 times more often by nonhomologous end joining-related deletions and insertions. Additionally, ZFN off-target activity based on binding mismatch sites at the locus of interest was detected in in vitro cleavage assays and also in chromosomal DNA isolated from treated cells. Therefore, the specificity of ZFN-induced ssODN-mediated gene repair needs to be improved, especially regarding clinical applications. Received 5 November 2009; accepted 14 December 2009; published online 12 January 2010. doi:10.1038/mt.2009.304

Introduction Genetic defects caused by a point mutation in a single gene can result in devastating health conditions, as exemplified by X-linked severe combined immunodeficiency. To compensate for this gene defect, functional gene copies have been introduced with viral vectors into the genome of autologous hematopoietic stem/precursor cells. After retransplantation, promising clinical success was observed1,2 however, some cases of leukemia several years after treatment correlated with vector integrations into specific chromosomal sites

of the hosts.3–5 Integration is an intrinsic property of the vectors’ molecular biology and is now, due to the clinical findings, under increased scrutiny.6–9 In an attempt to circumvent these difficulties, a nonviral gene repair strategy is being tested to correct the point mutation directly at the affected endogenous locus. According to such a scenario, cells of a patient are treated ex vivo by transfecting a single-stranded oligodeoxynucleotide (ssODN) as a repair matrix. Based on this “exogenous” information, the cellular repair machinery shall faithfully revert solely the mutation leaving unchanged the control elements of the endogenous chromosomal locus as well as all other sites of the genome. Previous work demonstrated that ssODNs alone can induce specific targeted correction of various chromosomal loci.10–14 Interestingly, experimental settings differing in, e.g., target loci, types of ODNs, transfection procedure, monotonically resulted in rather low repair rates.15 Furthermore, correction of a single-copy locus in the mammalian genome is hardly possible with ssODNs alone,11,16 but can be achieved with a concomitant defined DNA double-strand break (DSB) close to the mutation. Repair rates of ~0.2% were observed when the homing endonuclease I-SceI had been employed in targeting a single-copy reporter locus in a model cell line.17 Use of a homing endonuclease, however, is generally limited in that the canonical recognition site is not present in the human genome. Retargeting a homing endonuclease to any endogenous locus of interest is principally possible,18–20 but is likely to be limited to only a subset of all potentially interesting genomic locations. Chimeric zinc-finger nucleases (ZFNs) are an interesting alternative.21,22 They are formed by a nonspecific nuclease domain—derived from the type IIS restriction endonuclease FokI—linked to a DNA-binding module consisting of several zincfingers specific for the target sequence.23–25 Tailor-made ZFNs are obtainable from a commercial source but also from academic labs that developed publicly available platforms.26,27 The work presented here investigated the usefulness of a previously described pair of ZFNs28,29 in ssODN-mediated gene repair. After transfection of the respective nuclease expression vectors and a correction molecule, a mutated open reading frame of a stable single-copy enhanced green fluorescent protein (EGFP)derived reporter gene was to be reconstituted, and the repair rates

Correspondence: Klaus Schwarz, Institute for Transfusion Medicine, University of Ulm, Helmholtzstraße 10, D-89081 Ulm, Germany. E-mail: [email protected] Molecular Therapy vol. 18 no. 4, 743–753 apr. 2010

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Varied Outcomes of Gene Repair With ZFNs and ssDNA

the δEGFP open reading frame had to be reconstituted, and the LacZ and the EGFP open reading frames had to be fused in frame. Together with the functional ZFN pair GZF1-N + GZF3-N,29 the correcting ssODN 3-1corr resulted in rates of around 0.1% among living cells (Figure 2, lane 20), whereas no gene repair was observed with all other ZFN combinations, with 3-1scr, and in the mock samples (detection limit ~1 in 50,000). The intracellular expression levels of the four ZFNs differed (Supplementary Figure S1). Nevertheless, experiments titrating the amounts of expression vectors down to 1/8 revealed repair rates comparable to those in Figure 2 (data not shown). The double-stranded DNA (dsDNA) matrix restored the target locus at a rate comparable to that of the ssODN (Figure 2; lane 20 versus 21). The sequence analyses of loci from EGFP+ cells (targeted with ZFNs or I-SceI) confirmed faithful correction in 54–94% and in 92–100% of

were measured by fluorescence-activated cell sorting (FACS) analyses. Because the knowledge about unwanted side effects of gene targeting approaches is of central importance, here, the fidelity of the ssODN-mediated repair of an intended DSB was scrutinized by sequencing targeted sites from repair-positive and also repair-negative cells.

Results FACS analyses of repair rates To explore the possibility of stimulating ssODN-mediated gene repair by a ZFN-induced DSB, the previously described human model cell line 293/3-1 was used.28 These human embryonic kidney-293-derived cells harbor a stably integrated single-copy compound reporter locus (Figure 1). To allow FACS analyses of repair rates (as EGFP+ events), the missing 5′-terminus of CMV IE

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Figure 1 System outline: target locus of the 293/3-1 cells and the repair matrices for DSB repair. An integrated single copy of the plasmid pCMV.LacZs31δGFP (7,832 bp) constitutes the target locus. CMV IE: cytomegalovirus immediate early promoter/enhancer. LacZ: complete ORF encoding β-galactosidase. δEGFP: EGFP ORF with a 5′-deletion of 32 bp rendering EGFP nonfunctional. pA: SV40 polyadenylation signal. Gray wavy lines symbolize the flanking chromosomal sequences. The nucleotide sequence of the locus depicts the region where the 3′-terminus of the LacZ ORF is joined via a 42-bp fragment to the 5′-deleted EGFP ORF. For translational decoupling, stop codons (underlined) are introduced in frames +0 and +1. The binding sites for I-SceI and the ZFNs are highlighted with the color-coded jagged arrows pointing toward the respective cut sites. An AseI recognition site forms the 6-bp spacer region. ssODNs (all of sense orientation): primary structures of the repair and control matrices. Note that for 3-1corr, one version without and one with a BdT were available. 3-1c-s-BdT has a 5′-terminal 40-bp region homologous to the intact LacZ ORF whereas the remainder of the molecule—being identical to the 3′ part of 3-1scr—is unable to reconstitute the EGFP ORF. 3-1scr is composed of the nucleotides of 3-1corr but in a scrambled order. dsDNA matrix: the nucleotide sequence of this positive control covers the target site where both reporter ORFs are to be fused in frame. It has 2,697 repair-relevant base pairs: 5′ homology region of 1,950 bp (δLacZ ORF, 1,950 bp); repair segment of 41 bp (9-bp-stuffer; 32 bp reconstructing the 5′-terminus of EGFP ORF); 3′ homology region of 706 bp (688 bp specific for EGFP ORF, plus additional 18 bp abutting the EGFP ORF). Ovals mark the start codon of the cut with EGFP ORF. Scheme of pUC.Zgfp (5,845 bp): This plasmid harbors the dsDNA matrix. Note that this control was always used in its linearized form (E: EcoRI) with dephosphorylated 5′ termini to reduce direct circularization upon cell entry. Further sequence informations about the dsDNA matrix can be found in Supplementary Figure S7. Drawings are not to scale. BdT, biotinylated thymidine; bp, base pair; DSB, double-strand break; dsDNA, double-stranded DNA; EGFP, enhanced green fluorescent protein; ORF, open reading frame; ssODN, single-stranded oligodeoxynucleotide; ZFN, zinc-finger nuclease.

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the alleles with the ssODN and the dsDNA matrix, respectively (Supplementary Figure S2). With about 15% of corrected alleles carrying point mutation(s), the fidelity of targeted gene repair was ~10 times lower with the 112-mer ssODN compared to the dsDNA matrix. The control reactions with the dsDNA matrix showed about 10 times reduced levels of EGFP+ cells (Figure 2; lanes 5, 9, 13, 17). This background of fluorescent cells may have resulted from recombination events between the dsDNA matrix and the target locus without intentional DSB and/or from EGFP expression driven by a cryptic promoter located on the dsDNA matrix. In summary, the ssODN alone did not lead to detection of single-copy gene repair, but yielded EGFP+ cells when a DSB was concomitantly induced by the ZFNs. The rates were approximately two times higher than those obtained in the positive control with the homing endonuclease (Figure 2, lane 20 versus 24).

Fate of the ssODN during ZFN-induced gene repair Since the ssODN serves as information template during ssODN-mediated repair of a DSB induced by I-SceI,17 it was of

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Figure 2 Chromosomal repair rates. 293-3/1 cells were analyzed 7 days post-transfection. EGFP+ cells among the living (7-AAD−) cell population were counted by FACS. Mimicking the situation of an ex vivo protocol where corrected cells cannot necessarily be enriched from noncorrected ones, repair rates were related to all living cells, i.e., no normalization was calculated. Transfection quality controls at 5 hours postnucleofection (see Supplementary Materials and Methods) resulted in nine valid independent experiments the data of which are presented in box plot format. Black bar: median; right edge of the gray box: 75% quartile; left edge of the box: 25% quartile; the horizontal line connects the minimum value with the maximum value. MOCK: nuclease-negative control with plasmid pRK5, the backbone-only version of the expression plasmids. In the mock samples for the matrices, cells received a calcium phosphate-precipitate with no DNA added. Statistical significance (P = 0.05) was tested with the two-tailed Wilcoxon matched pair signed-rank test: Null hypothesis not rejected (*), rejected (**, ***). dsDNA, doublestranded DNA; EGFP, enhanced green fluorescent protein; FACS, fluorescence-activated cell sorting.

Molecular Therapy vol. 18 no. 4 apr. 2010

interest whether there is also no ssODN incorporation during ZFN-initiated gene repair. To this end, biotinylated ssODNs were used to then recover genomic DNA (gDNA) fragments in case they had obtained a biotin label during the experiment. Fragments enriched by a capture assay were amplified by PCR, and the results of two independent experiments are presented (Figure 3). To monitor functionality of capturing and amplification, all reactions, except the water controls, were spiked with defined amounts of an internal control template. The titration reactions (Figure 3b; lanes 5–7) proved that internal control and the test fragment (Figure 3a) could be coamplified and that captured gDNA fragments were detectable at least down to 20% of the total input material. Note that the relative product amounts varied between different repeats. The reasons for this are unknown. In experiment I, no PCR signal was observed in 3-1corr-biotinylated thymidine (BdT)-treated cells (Figure 3b; lanes 3, 4), and thus no incorporation was detected. This led to the conclusion that in concert with ZFN-induced breaks, the ssODN serves as an information template as described for I-SceI.17 No locus-specific signal was detected in the negative controls (Figure 3b; lanes 1, 2). In experiment II, however, a signal appeared in the sample from gene repair-positive cells (Figure 3b; lane 4). Thus, a fraction (below 20%) of the gDNA fragments from EGFP+ cells carried a biotin label. At this point, it was unclear whether the biotinylated 3-1corr-BdT was a fully ligated integral part of the locus’ top strand or whether the ssODN merely trapped gDNA fragments via hybridization. To distinguish between these possibilities, the capture assay was repeated (a second aliquot from experiment II) with an additional NaOH treatment to remove not fully ligated DNA strands. Upon NaOH treatment before PCR amplification, a twofold reduction of the signal would be expected due to the presence of only half of the template DNA strands. Because the signal strength was ~2/3 of that typical for strand removal (Figure 3c), it was concluded that the majority of the ssODNs had been ligated into the chromosomal DNA strand at their 5′ and their 3′ end. It must be noted that experiment II was more efficient than experiment I, because the repair rate was enhanced (0.18% versus 0.11%), and the cell density at the time of harvest was higher. From this it was presumed that cellular ssODN concentrations were elevated, and more cells duplicated after the transfection procedure. It thus seemed likely that the signal arose from targeted loci which first had undergone successful ZFN-induced DSB repair without ssODN incorporation, but thereafter interacted with the now perfectly matching 3-1corr-BdT still present intracellularly during a subsequent S phase. Sequence analyses of these captured subcloned alleles confirmed faithful ssODN-mediated gene repair (data not shown).

Biotin-based assay analyzing unwanted events at the targeted site Capture assays were repeated with ~120 times more input gDNA from sorted EGFP− cells to allow detection also of rare unwanted events. Figure 4a,b schematically depict the target locus with the PCR primers and products, and the effect of the NaOH treatment, respectively. In general, the increased amounts of input alleles resulted in pronounced ladders of PCR products (Figure 4c). Each sample showed a distinct pattern because—most likely—each 745



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Figure 3 Capture assay analyzing the fate of an ssODN during targeted gene correction. The 292-bp fragment (gray arrow head) amplified with primers gfpM and gfp7 from the internal control IC17 monitored the functionality of the PCR and the completeness of the NaOH-mediated bottom strand removal. (a) Schemes of the biotinylated test fragment TF (top) and of a biotinylated target locus-specific gDNA fragment (bottom). TF served as locus-specific positive control for the capturing step also allowing the sensitivity of the PCR-based detection step to be assessed. TF was generated by hybridizing 3-1corr-BdT to the bottom strand of a PCR product (primer pair: #48-LacZ-TC/pabox4; template: corrected locus) followed by a oneround elongation. The 452-bp product arises from TF based on its bottom strand. Drawings are not to scale. (b) PCR results of capture assays with gDNAs from sorted EGFP– and EGFP+ cells. To separate the PCR products in 1.5% agarose gels, 10% of the reaction volumes were loaded per lane. Data from two independent experiments are presented (input: 200 cells (I), 500 cells (II)). The indicated percentages for IC and TF compare their molecule numbers with the numbers of the target alleles (which are equivalent to the number of cells). (c) Results of the capture assay probing whether the biotinylated ssODN is fully ligated into the top strand. From experiment II, a second sample of 500 EGFP+ cells transfected with 3-1corr-BdT was split into two aliquots one of which was NaOH treated before the PCR. M: 100-bp DNA Ladder. Numbers next to the lanes M indicate fragment lengths in bp. The gels were stained with ethidium bromide and are shown in reverse contrast. bp, base pair; EGFP, enhanced green fluorescent protein; gDNA, genomic DNA; IC, internal control; ssODN, single-stranded oligodeoxynucleotide; TF, test fragment.

product was generated uniquely from one independent event during the repair experiment. The original target locus itself gives rise to a 465-base pair (bp) fragment. The apparent sizes of most of the amplification products were >465 bp. In the case of 3-1corrBdT, treatment with NaOH to select for fully ligated gDNA fragments resulted in relatively increased amounts of the products with lengths >465 bp. Sequence analyses of subcloned PCR products revealed that ssODNs had been inserted between the termini of the ZFN-induced DSB, in some cases together with unrelated chromosomal sequences (Figure 4d; target locus alignments for all sequences analyzed are compiled in Supplementary Figure S3). Nonhomologous end joining (NHEJ)-modified and original target loci found in 3-1corr-BdT samples were not observed after the NaOH treatment. This showed that the bottom strands of those target loci were attached to the beads only via duplex formation with 3-1corr-BdT which was either not ligated at both termini or ligated at only one terminus. To estimate the frequency of unwanted insertions at the intended DSB, the amounts of captured gDNA fragments were 746

assessed by evaluating a defined PCR product generated from a region downstream of the ZFN target site (primer pair gfp1/gfp7; Supplementary Figure S4). Assays were carried out with the NaOH treatment to amplify preferably completely ligated fragments. Because a top strand carrying 3-1corr-BdT with a 5′ nick and a 3′ end elongated at least ~200 bp could also be amplified, the results from 3-1c-s-BdT represent better the numbers of fully ligated ssODNs. Several repeated assays with gDNAs from EGFP− cells showed that roughly 1% of all cells carried biotinylated ssODN sequences inserted into the ZFN-induced DSB (Supplementary Figure S4). Compared with the repair rate of 0.16%, these unwanted insertions are about six times more frequent than the intended repair event.

Biotin-independent assay analyzing unwanted events at the targeted site As mentioned above, ssODNs were sometimes integrated into the DSB site together with chromosomal DNA. This led to the question whether ZFN-targeted loci might be modified also without www.moleculartherapy.org vol. 18 no. 4 apr. 2010


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Figure 4 Analyses of unwanted events at the targeted site in EGFP− cells. (a–d) Capture assay based analyses. (a) Scheme of a target locusspecific gDNA fragment carrying an integrated part of a biotinylated ssODN at the DSB site. Black line: top strand; gray line: bottom strand. Primers #48-LacZ-TC and gfp7 encompass the repair site resulting in fragments of different lengths depending on the respective DSB rejoining. The original target locus yields a 465-bp long fragment. The primer pair gfp1/gfp7 amplifies a region of the EGFP ORF 42-bp downstream of the GZF1-N binding site assessing the total number of captured biotinylated alleles (see also Supplementary Figure S4). (b) Scheme depicting the effect of the NaOH treatment on the PCR amplification when the targeted loci contain a 3′-ligated ssODN. All drawings are not to scale. (c) PCR result of capture assays (primer pair #48-LacZ-TC/gfp7). For each sample, 250 ng of gDNA (corresponding to an estimated number of ~28,000 targeted alleles) were utilized. TF monitored the removal of the bottom strand. Ten percentage of the PCR products were analyzed. (d) Summary of sequence results from c. The numbers on top of the columns refer to the total numbers (Σ) of sequences which were scored as independently generated. (e–f) Biotin-independent analyses. (e) Gel-electrophoretic separation of products directly amplified from targeted loci. For each sample, 100 ng (~11,100 target alleles) were employed. Digestions of the gDNAs were carried out with AseI to reduce background generated from unmodified target loci. DraI (also cutting an AT-rich hexamer sequence) was used to control the AseI-specific enrichment of modified loci, and to monitor the ensuing PCR with primer pair #48-LacZ-TC/gfp7 in the presence of high numbers of gDNA fragments. (f) Summary of the sequence results from samples of e. The numbers on top of the columns refer to the total numbers (Σ) of sequences which were scored as independently generated. All gel pictures are presented in reverse contrast. M: 100-bp DNA ladder. Numbers next to the marker lane M indicate fragment lengths in bp. bp, base pair; DSB, double-strand break; EGFP, enhanced green fluorescent protein; gDNA, genomic DNA; ORF, open reading frame; ssODN, single-stranded oligodeoxynucleotide.


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Figure 5 In vitro assay of zinc-finger nuclease activity. (a) Digestion of the circular target locus plasmid pCMV.LacZs31δGFP by in vitro-made ZFNs. Reaction products were separated in a 0.8% agarose gel and stained with ethidium bromide. The picture is shown in reverse contrast. As positive control for linearization (lane 11), pCMV.LacZs31δGFP was digested with BamHI. L: 1-kb DNA ladder. Numbers to the right of the marker lane L indicate fragment lengths in kb. (b) Scheme of the artificial linear substrate with both 5′-DIG top and 5′-DIG bottom label. Numbers indicate the lengths (nts) of the expected single-stranded digestion products. Canonical binding sites are uniformly highlighted and the ZFN name is written with standard letters. Nonoptimal binding sites are named with italic letters, matches are highlighted. The second GZF1-N-specific site (nts 292–300 of the EGFP ORF) has been located by in silico analyses. The jagged triangles point to the cleavage positions. The gray thick lines refer to the respective spacers with boxed numbers indicating their lengths in bp. Drawing is not to scale. (c) Digestion of a linear DIG-labeled substrate. The reaction products rendered single-stranded were separated in a 1.5% agarose gel and transferred onto membranes. Labeled DNA strands were visualized with a DIG-specific antibody coupled to alkaline phosphatase. 5′-DIG top, 5′-DIG bottom, 5′-DIG top + bottom: PCR products carrying a DIG-label either in the top strand, the bottom strand, or in both. Numbers to the left indicate the lengths (nts) of the original DNA strands (black arrowhead) and the corresponding products (gray arrowheads). Note that these unspecific digestion products in lanes 4, 8, 9, 12, and 13 were observed in 3 out of a total of 5 independent experiments. The reason for this is unknown. bp, base pair; DIG, digoxigenin; nt, nucleotide; ZFN, zinc-finger nuclease.

the participation of the ssODN. Thereto, gDNA samples from EGFP− cells were analyzed omitting the bead-capturing. Targeted loci were amplified either directly or after digestion with AseI to select against the original target locus alleles. DraI digested gDNAs served as a control. Gel-electrophoretic analysis (Figure 4e) showed for all samples the target locus-specific 465-bp PCR product. Only after AseI digestion, lower signal intensities were observed in the untransfected samples as compared to the transfected ones (Figure 4e; lanes 1–6) due to a diminished numbers of original target alleles. In all transfection samples, smears representing products of lengths >465 bp were observed. Subcloning and sequencing confirmed that namely after AseI-related target allele reduction, the majority of amplified sequences had undergone NHEJ-related sequence modifications (Figure 4f; all sequences analyzed are presented in Supplementary Figure S5). In addition, 748

integrations of (i) ssODNs, (ii) human and nonhuman chromosomal sequences, and (iii) parts of the backbone of the ZFN expression vectors were detected above a background of unmodified target loci. Finally, a 27-bp fragment was found duplicated ~50 bp upstream of the unchanged ZFNs’-binding sites resulting in a direct head-to-tail repeat (Supplementary Figure S5). It is unclear whether here a ZFN-induced DNA break was involved.

Characterization of ZFN off-target activity The ZFNs used in this study, GZF1-N and GZF3-N, contain reengineered nuclease domains that were shown to decrease ZFNassociated toxicity significantly.29 In particular, these ZFNs harbor mutations in the dimer interface of the FokI nuclease domains (I538V and I499A, respectively), which decrease the dimerization energy, but maintain nuclease activity comparable to wild-type www.moleculartherapy.org vol. 18 no. 4 apr. 2010


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study—cannot be excluded and is likely strongly dependent on (i) the homodimerization property of the respective FokI domain and (ii) the DNA-binding specificity.

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DNA inserted 5.

Figure 6 Recapitulating gene repair experiment outcomes. The boxes represent the different categories of alleles found in the cell population. The categories are listed in descending order according to their frequencies. “>>“: many more alleles as compared to the following category. Note that not all alleles can be detected due to failures in, e.g., bead-capturing, PCR amplification, subcloning. Thus, the rankings are not based on absolute numbers of the different alleles detected, but describe the findings semiquantitatively. DSB, double-strand break.

levels.29 The nonfunctional GZF1-KO and GZF3-KO served as negative controls for DSB-induced repair. Evaluation of the ZFNs activities in vitro confirmed that they cleaved the intended target site on a plasmid encoding the transgene locus (Figure 5a). In a more sensitive in vitro cleavage assay involving a digoxigenin (DIG) end-labeled PCR fragment (Figure 5b), unspecific offtarget activities at two sites were detectable with GZF1-N alone (Figure 5c, lane 4) and, to a lesser extent, in conjunction with GZF3-KO or GZF3-N (lanes 8, 9). These off-target activities were clearly dependent on the catalytic activity of GZF1-N as no offtarget cleavage activity was observed for GZF1-KO. To confirm the position of the off-target site located in silico in the EGFP open reading frame, an experiment was performed with substrate fragments carrying only one 5′-DIG label. Based on the finding that ZFNs generate 5′-overhangs, as does the wild-type FokI enzyme,30 a 514-nucleotide and a 496-nucleotide strand were predicted for the cut in the top and the bottom strand, respectively. The data shown in Figure 5c (lanes 12, 13) corroborated this prediction, also proving that the ZFNs indeed generated a DSB. Taken together, these results show that GZF1-N and GZF3-N cleave their target site efficiently in vitro and they also point toward some offtarget activity of GZF1-N. Interestingly, this off-target activity has already been observed with an earlier version of this ZFN carrying the wild-type FokI nuclease domain.31 To explore whether these activities are also detectable in cellula, cells were transfected with only one of the two ZFNs. After AseI or, for the second GZF1-N specific site, MboII digestion of the gDNAs, targeted loci were amplified by PCR, redigested, and undigestable products were subcloned and sequenced. Note that technical limitations did not allow for enrichment of possibly modified alleles from the second ZFN target site, and thus no respective in cellula data are available. About one NHEJ-derived modification per ~15,000 alleles was observed at the regular ZFN site for GZF1-N alone (Supplementary Figure S6). Without further analyzing which of the three proposed configurations was involved during homodimeric interaction of GZF1-N, the finding demonstrates that as soon as one ZFN protein has bound to its canonical 9-mer recognition site, the homomeric partner can bind functionally even to a mismatched binding site. Because similar binding site variations are present for a GZF3-N homodimer (Figure 5b), off-target activity—even though not detected in this Molecular Therapy vol. 18 no. 4 apr. 2010

The ZFN technology promises to allow the introduction of a DSB at almost any predefined position in mammalian genomes. To explore the usefulness of ZFNs in targeted single-copy gene repair mediated by ssODNs, a previously characterized ZFN pair was scrutinized for this purpose. A special focus was put on the fidelity of gene repair at the targeted site.

ZFN-induced DSB repair with an ssODN The 112-mer 3-1corr was designed to replace a chromosomal 45-bp segment. The successful gene repair events demonstrated that a 40-nucleotide homology arm on either side of the molecule was sufficient for exchanging the genomic segment during DSB repair via a template mechanism. The relatively low repair rate of 0.16% achieved here can hardly be compared to rates from differently designed gene repair experiments, because the experimental outcome is strongly dependent on a variety of parameters such as delivery technology,32 chromosomal locus, cell type, and repair matrix topology. Compared to the positive control experiment with the dsDNA matrix, the repair rates were similar (Figure 2), but it is worth noting that the numbers of dsDNA matrix molecules offered during transfection were only ~1/50. Underlying reasons for the less efficient ssODN-mediated repair might be that (i) the active concentration of the 112-mer at the target site was reduced due to, e.g., nuclease activities or (ii) the repair pathway utilizing the dsDNA matrix is generally more efficient. Repair rates with 3-1corr measured in the I-SceI-based control experiments were only ~50% of those with the ZFN pair. Targeting experiments where the nucleases were used without repair matrices revealed that I-SceI digestion, as compared to the ZFNs, resulted in lower numbers of mostly intact DNA termini (data not shown). Thus, it was presumed that the interaction between the ssODN and the I-SceI-targeted site, which depends on sufficient homology, was reduced and consequently, the repair rates were diminished. Targeted gene repair experiments rarely achieve rates above 1%,12,13,29,33 with the notable exception of using integrase-deficient lentiviral vectors where—depending on the cell type—repair rates in the double-digit percent range have been reported.32,34 It is conceivable that properties inherent to viral systems, such as an efficient delivery of the nuclease and matrix vectors to the nucleus, enable these relatively high-correction rates. The fact, however, that integrase-deficient lentiviral vectors randomly integrate into the genome of initially successfully transduced human embryonic kidney-293T and human CD34+ cells at rates of ~1%34 and ~0.04%,32 respectively, complicates the development of safe protocols. Transient transfections of an expression vector encoding, e.g., a fluorescent reporter protein yield up to almost 100% positive cells in many model systems. The generally low repair rates on the other hand remain unsatisfactory with the reasons not fully understood. In contrast to expressing a reporter gene, more complex biochemical machineries are likely to be required for successful gene 749


correction. Two defined nuclease proteins need to concomitantly be generated resulting in concentrations which allow formation of an active heterodimer at the nuclear target site. Furthermore, the repair matrix—the third player to be applied from outside of the cell—has to be present. Only when such a multiple-molecule delivery has been achieved at the chromosomal site, nuclear factors can take over to carry out the intended repair process. From research especially in cancer and stem cell biology, however, evidence is accumulating that not all cells of a certain population show the same gene expression pattern (for a review see Huang35). Even though based on the same gene regulatory network, cells are distributed through a space of different states rendering only few of them responsive to a given stimulus, here precise DSB-induced gene repair; this probably is one important reason for the difficulties in achieving higher rates of faithful targeted gene repair.

Unwanted events at the targeted site The experiments examining the fidelity of targeted gene repair revealed increased numbers of point mutations at the target site when ssODN instead of a dsDNA repair matrix was used. One explanation is the accumulation of faulty products during the synthesis of the 112-mer ssODN and the inability of the gel purification step to remove these point-mutated full-length molecules from those with the correct sequence. Alternatively, a reduced fidelity might also be observed if the cellular DNA repair process utilizing the ssODN is more error-prone. Applications that require high-cell numbers necessitate expansion of the cell population of interest. However, this may be problematic because genetic changes, such as trisomy and copy number variation, might accumulate during prolonged culturing, as recently shown for murine embryonic stem cells which till then were presumed to be karyotypically rather stable.36,37 To minimize or even avoid such a risk, one would like to repair the gene of interest, if not in all then at least in the majority of the targeted cells, as pictured in Figure 6a in the form of a small inequality. However, in the gene repair study presented, extensive sequence analyses (Figure 4, Supplementary Figures S3 and S5) revealed a complex allele distribution (Figure 6b). The favorite outcome—a repaired allele— only occupies the 4th position in this ranking. The inequality shows that most of the targeted loci remained unchanged, consistent with the low repair rate. The additional categories demonstrate that a substantial number of targeting events yielded unwanted locus modifications. NHEJ constituted the major cellular repair pathway leading to unwanted modifications (Figure 4f). The next group comprises alleles where ssODN sequences were added between the termini of the intended DSB, in ~70% of the cases via microhomology (Supplementary Figures S3 and S5). It is likely that this type of modification took the ranking’s 3rd position due to high amounts of transfected molecules. Compared to the rates of faithful repair, the approximately sixfold higher incidence of ssODN addition emphasized that in the case of a DSB, the cellular machinery clearly opts for inserting pieces of DNA between the termini. The relatively high number of insertions was likely detectable by utilizing the described sensitive capture assay which allows the recovery of single events from bulk gDNAs. The strong recombinogenic nature of DNA was recently also reported from experiments aimed at generating murine-induced pluripotent stem cells. Even though 750


in that case, double-stranded circular plasmids were transfected without concomitant DSB induction, ~70% of all clones generated carried integrated plasmid sequences.38 The finding of chromosomal fragments cointegrated with the ssODN underlines the dynamics of genomic recombination. It is worth noting that ~20% of all alleles carrying part(s) of the ssODN could not completely be sequenced (Supplementary Materials and Methods) due to hard stops which in all likelihood were caused by secondary structures. It is unknown whether such DNA segments might induce genetic instability in the affected cells. The last group of sequence additions represents chromosomal fragments without ssODNs. These fragments might be derived from physiological DSBs which happened to occur during the targeted gene repair. In two independent experiments, sequences derived from Bos taurus were identified in the cellular genome (Supplementary Figures S3 and S5). These DNA fragments were likely culture medium-born, whereas it is unclear whether they were introduced into the target cells during the nucleofection or the calcium phosphate-mediated step. In any case, this underscores the high propensity for genetic recombination accepting almost any nucleic acid piece. To avoid such events during targeted gene repair, culture conditions should be chemically defined to possibly exclude any contaminating DNA molecule. By analyzing unwanted events, it also became apparent that 3-1corr-BdT—even though not stabilized against nuclease-mediated degradation—was still present at the targeted site of EGFP− cells 2-days post-transfection (Figure 4d). This discovery of prolonged ssODN activity was possible, because 3-1corr-BdT trapped the bottom strand of an original or of an NHEJ-modified locus by DNA duplex formation. Of note, the NaOH treatment proved that 3-1corr-BdT was not fully ligated into the top strand. Such trapping was not observed with the control ssODN 3-1c-s-BdT even though it has homology to the target locus albeit confined to its 5′ half. Therefore, it likely is the 3′ terminus of an ssODN, possibly also priming DNA synthesis, which is responsible for this effect. In summary, homology-guided interaction of the ssODN with a chromosomal site occurred during extended time periods, and this corroborates the conclusion drawn from the data of Figure 3c.

Revisiting critical ZFN parameters To minimize unwanted off-target activity across the entire human genome, the ZFNs should be present in the cell nucleus only transiently and at concentrations as low as possible. In this context, it is interesting to note that neither the codon usage of GZF1-N and GZF3-N was humanized nor were these proteins provided with an additional nuclear localization signal. The expression levels might thus have been suboptimal correlating with the low repair rate. However, as shown previously chimeric ZFNs are able to localize readily to the nucleus even without an nuclear localization signal,29,39 and because the repair rates were unchanged with only 1/8 of the amounts of the expression vectors offered, the DSBinducing agents were not a limiting factor. The sensitive in vitro assay to assess ZFN activities revealed unspecific activity by GZF1-N (Figure 5). One additional cleavage site was identified on the short substrate based on fragment size (Figure 5c) and target sequence homology (Figure 5b). Furthermore, GZF1-N was able to cleave the actual target site as a homodimer. More importantly, these results were confirmed in www.moleculartherapy.org vol. 18 no. 4 apr. 2010


c ellula, at a frequency of about 1 in ~15,000 alleles. Mani et al.40 speculated that ZFN-associated cytotoxicity is probably due to cleavage at secondary degenerate sites positioned in an inverted orientation. The finding here that one ZFN contacted its canonical 9-mer recognition site and its partner bound to a partially mismatched site proved the authors’ speculation right. Interesting in this context, BLASTN searches across the haploid human genome (Ensembl Genome Browser) revealed that GZF1-N and GZF3-N could potentially interact with ~24,000 and ~12,000 additional perfect chromosomal binding sites, respectively (data no shown). Thus, off-target events are likely to happen, if heterodimer formation is not strictly required for activity and/or if one or both ZFN subunits are active when bound to an imperfect target site. These findings hence re-emphasize that all three domains of a ZFN have to be optimized to keep off-target activity at a minimal level. Previous site-by-site analyses suggested that—as opposed to modular assembly which was used here—a context-sensitive selection method to generate the DNA-binding domain may result in ZFNs with higher activity and lower toxicity.41,42 The interdomain linker used here was 6-amino acid long and therefore allowed cleavage of DNA at sites that do not contain the canonical 6-bp spacer between the target subsites (Figure 5b). The use of a 4-amino acid linker should substantially reduce such off-target activity.43 Finally, re-engineered nuclease domains that prevent homodimerization should be used.29,44 Taken together, this study identified several major obstacles to efficient and highly specific ZFN-induced gene repair via ssODNs: (i) The quality of ssODNs must be thoroughly tested to avoid introduction of unwanted mutations due to faulty products. This will likely be challenging because gel purification, capillary electrophoresis, and matrix-assisted laser desorption/ionization-time of flight analyses are all limited in their performance. (ii) Even unmodified ssODNs might be active inside cells for prolonged times leading possibly also to interactions with unrelated sites of similar sequences elsewhere in the human genome. (iii) The DSB, which is required for targeted modification of a single-copy locus, results in considerably more ssODN insertion events than precise repair reactions. This disproportion is one reason for the low rates. (iv) Only a few nucleotides of homology suffice during sealing of the DSB termini with ssODN sequences. Because DSBs also occur under natural conditions elsewhere in a genome, microhomology-based joining of those breaks with various DNA fragments counteracts highly specific genome modifications. (v) As long as ZFNs also act nonspecifically, the rates of unwanted events are increased. Thus, further improvement of these chimeric nucleases is of paramount importance to increase the specificity of the interaction with the target site. Finally, it is inevitable to analyze gDNAs of the targeted cells at high resolution and sensitivity, and—in case of unwanted modifications—to interpret carefully the data in terms of their biological significance.

Materials and Methods Detailed descriptions of the materials, methods, and software are compiled in Supplementary Materials and Methods. Plasmids and oligodeoxynucleotides. The target locus with plasmid pCMV. LacZs31δGFP and the repair matrices are introduced in Figure 1. ZFNs GZF1-N, GZF3-N, GZF1-KO, and GZF3-KO were encoded by plasmids pRK5.GZF1-N.I538V, pRK5.GZF3-N.I499A, pRK5.GZF1-N.D450AG, and pRK5.GZF3-N.D450AG, respectively. pRK5.LHA-Sce128 was the Molecular Therapy vol. 18 no. 4 apr. 2010


expression vector for the homing endonuclease I-SceI (positive control for the effect of DSB induction). The ssODNs 3-1corr (112-mer; gel-purified 98%) and 3-1scr (112-mer; gel-purified 99%) were received from Eurofins MWG (Ebersberg, Germany), the biotinylated versions 3-1corr-BdT (112mer; gel-purified 96%) and 3-1c-s-BdT (113-mer; gel-purified 85%) were obtained from Eurogentec S.A(Seraing, Belgium). Cell culture. The human embryonic kidney-293-derived transgenic 293/3-1

cell line28 was expanded in DMEM (Gibco Invitrogen, Karlsruhe, Germany) supplemented with 10% fetal calf serum (PAA, Pasching, Austria), 2 mmol/l l-glutamine (Gibco Invitrogen), and 0.5 mg of Geneticin (G-418; Gibco Invitrogen) per ml medium. In preparation for each experiment, cells were reactivated from frozen aliquots and maintained in medium without Geneticin.

Transfections. Each experiment employing a nuclease and a repair matrix was carried out as an individual sample according to a two-step transfection protocol. Per 1.5 × 106 cells, a total of 5 µg of the nuclease expression plasmids was nucleofected according to the manufacturer’s instructions (Amaxa Nucleofector Kit V (Lonza Cologne AG, Cologne, Germany)). Two hours later, 0.5 µg of the respective repair matrix was transfected via calcium phosphate-mediated co-precipitation. For each nuclease combination, four different calcium phosphate co-precipitates were transfected each employing a 24-well culture aliquot: mock, 3-1scr, 3-1corr, and dsDNA matrix. On day 2, cells were passaged from 24-wells to 6-wells. In case of the 2/9 experiments where also the expression of the nucleases was evaluated via intracellular influenza hemagglutinin tag staining, cells were passaged on day 1. To study the fate of ssODNs, 7.5 µg of biotinylated ssODNs were co- precipitated onto cells combined from three nucleofection cuvettes. FACS analyses and cell sorting. Transfection rates and repair efficiencies were evaluated with a BD FACSort Flow Cytometer System (Becton Dickinson, Heidelberg, Germany) on day 7 utilizing 50,000 total events. Cell sorting was carried out with a BD FACSAria Cell Sorting System (Becton Dickinson) on day 2 (bead-capturing) or on day 7 (assessing the fidelity of repair). Capture assay17 and analysis of biotinylated DNA fragments. Biotinylated DNA fragments were captured on streptavidin-coated beads (magnetic Streptavidin-Beads MyOne C1; Invitrogen, Karlsruhe, Germany) and then detected by PCR using the Taq PCR Polymerase System (QIAGEN, Hilden, Germany) and the primer pairs #48-LacZ-TC/gfp7 or gfp1/gfp7. For each sample, triplicates of 250 cells each (experiment I) or 550 cells each (experiment II) were sorted directly into a lysis buffer. The presence of DNA in the samples was tested by PCR using 50-cell aliquots. The remainders of the DNA-containing samples (experiment I: 200 cells; experiment II: 500 cells) were stored at −20 °C until capture assay. The Wizard Genomic DNA Purification Kit (Promega, Mannheim, Germany) was used to prepare the gDNAs from sorted EGFP− cells and 250 ng of digested gDNA were incubated with streptavidin-coated beads. In order to remove the DNA bottom strands and not fully ligated parts of the top strands, samples were treated with NaOH before PCR. Captured fragments were amplified by PCR and separated in agarose gels stained with ethidium bromide. PCR amplification of targeted loci from EGFP− cells without capturing.

For PCR analyses, gDNAs were either left undigested or were digested with AseI or DraI (New England Biolabs, Frankfurt am Main). Hundred nanogram of gDNA (equivalent to ~11,100 target alleles) served as PCR template using Stratagene PfuTurbo Hotstart DNA polymerase (Agilent Technologies Sales & Services & Co. KG, Waldbronn, Germany) and primer pair #48-LacZ-TC/gfp7. PCR products were separated in agarose gels stained with ethidium bromide. In vitro cleavage assay. DIG-labeled fragments for the in vitro cleavage

assays were generated by PCR with primers #48-LacZ-TC and pabox4 ± 5′-DIG-label. For producing the ZFNs, the respective SalI-linearized

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plasmid was incubated in TNT SP6 High-Yield Protein Expression System lysate (Promega). The in vitro cleavage assays (final volume: 10 µl) consisting of 1 µl of freshly generated ZFNs, 1× NEB4 buffer (New England Biolabs), 50 mmol/l NaCl (Merck KGaA, Darmstadt, Germany), and 250 ng of plasmid DNA or 10 ng of DIG-labeled PCR fragment were carried out at 37 °C for 1 h. For gel analysis of products from reactions with plasmid DNA, 1 µl of each digestion reaction was run on an agarose gel. In case of the digested DIG-labeled fragments, 1 µl of the reaction was mixed with 9 µl of formamide gel loading buffer II (Ambion; Applied Biosystems). The DNA fragments were heat-denatured, separated in an agarose gel, and transferred to a positively charged nylon membrane (Roche). The DIGlabeled fragments were detected with Anti-Digoxigenin-AP Fab fragments (Roche, Mannheim, Germany). The characterization of ZFN off-target activity in cellula is described in Supplementary Materials and Methods. Sequence analyses. For sequence analyses, PCR products were subcloned and inserts were sequenced (BigDye Terminator v1.1; ABI Prism 3100 genetic analyzer; Applied Biosystems). To group the sequences into the predefined categories and calculate their respective frequencies (Figure 4 and Supplementary Figure S2), the following general assumptions were made: any sequence which deviated after targeted gene repair attempts from the sequence of the original locus was assumed to be a unique event, and therefore, only the number of different types of sequences was used for calculation rather than counting each sequenced bacterial clone. However, concerning the original target locus, each sequenced bacterial clone was counted, because target locus alleles constituted the majority of template fragments and were thus likely amplified independently. To arrive at the percentages for the categories, the numbers of the respective independent sequences were divided by the number of all independent events of the respective experimental sample.

Supplementary Material Figure S1. Intracellular staining of transiently expressed zinc-finger nucleases. Figure S2. Quality control appraising the gene repair fidelity at the corrected target site of the ZFN pair. Figure S3. Biotin-based assay analyzing unwanted events at the targeted site: Compilation of all captured gDNA fragments analyzed. Figure S4. Quantifying frequencies of unwanted insertions of biotinylated ssODNs in EGFP-negative cells. Figure S5. Biotin-independent assay analyzing unwanted events at the targeted site: Compilation of all gDNA fragments analyzed. Figure S6. Analysis of in cellula ZFN off-target events at the target locus: Compilation of sequence modifications. Figure S7. Sequence information about the dsDNA matrix. Supplementary Materials and Methods.

ACKNOWLEDGMENTS We thank Ingrid Peter for her work in the early phase of the project. We are grateful to Claudia Friesen for a helpful hint concerning the cell lysis buffer in the capture assay protocol. We thank Ulrike Stötzner for her technical help, and Constanze Gruber, Katja Heinrich, Marina Jans, and Tatjana Kersten for their assistance in sequencing. We thank Doris Niewolik for her critical reading of the manuscript. This work was supported by the Institute for Clinical Transfusion Medicine and Immunogenetics Ulm, and by EU-Grant LSHB-CT2006-037783 ZNIP to K.S. and T.C.

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