TY - JOUR
AU1 - Collin, Joseph
AU2 - Lako, Majlinda
AB - Abstract Human pluripotent stem cells (hPSCs) encompassing human embryonic stem cells and human induced pluripotent stem cells (hiPSCs) have a wide appeal for numerous basic biology studies and for therapeutic applications because of their potential to give rise to almost any cell type in the human body and immense ability to self‐renew. Much attention in the stem cell field is focused toward the study of gene‐based anomalies relating to the causative affects of human disease and their correction with the potential for patient‐specific therapies using gene corrected hiPSCs. Therefore, the genetic manipulation of stem cells is clearly important for the development of future medicine. Although successful targeted genetic engineering in hPSCs has been reported, these cases are surprisingly few because of inherent technical limitations with the methods used. The development of more robust and efficient means by which to achieve specific genomic modifications in hPSCs has far reaching implications for stem cell research and its applications. Recent proof‐of‐principle reports have shown that genetic alterations with minimal toxicity are now possible through the use of zinc finger nucleases (ZFNs) and the inherent DNA repair mechanisms within the cell. In light of recent comprehensive reviews that highlight the applications, methodologies, and prospects of ZFNs, this article focuses on the application of ZFNs to stem cell biology, discussing the published work to date, potential problems, and future uses for this technology both experimentally and therapeutically. Zinc finger nucleases, Human pluripotent stem cells, Gene targeting Targeted Gene Editing in hPSCs Various approaches have been used to alter the genome of human pluripotent stem cells (hPSCs) [1]. Elucidation of gene function and mutation in human cells has routinely been accomplished by constitutive or inducible transgene expression and downregulation of gene expression, commonly by RNA interference (RNAi). These approaches are useful but the data may not accurately reflect the endogenous setting or function of the gene. For example, expression of an integrated transgene can be unpredictable over time due to atypical epigenetic changes occurring near its site of integration [2]. There is also the complication of multiple integrations of the transgene per cell and disruption or activation of other genes, creating a nonisogenic experimental setting. A targeted approach to genetic examination avoids these effects and allows for the specific editing of the genome providing a truer representation of genetic behavior in its native environment. Homologous recombination (HR)‐mediated gene targeting uses HR repair mechanism of a cell by delivering a DNA template with long regions of homology to the target locus. This technique has been routinely used for successful and efficient gene targeting in mouse embryonic stem cell (ESC) (with a HR rate of one in 103 cells [3]) to knock genes in and out and generate transgenic lines, which have been important for discovering gene function [4–6]. This method has also been demonstrated in hPSCs. However, following the first report of gene targeting in human ESCs (hESCs) by Thomson et al. in 1998 [7], only a few other examples of its success have been demonstrated to date [8–14]. Despite these reports demonstrating the viability of this approach, they also illustrate the technical difficulties involved, which results in a low HR rate of less than 10−6. Thus, recent publications demonstrating increased HR efficiency at specific sites using zinc finger nucleases (ZFNs) in many cell types [15–17], including hPSCs [18–22], holds new promise for the stem cell research field. Zinc Finger Nucleases ZFNs [23, 24] are modular enzymes designed to recognize a specific DNA sequence by combining C2H2 zinc finger proteins (ZFPs) [25] into a customized array [26–29]. This zinc finger domain is linked to the nonspecific FokI nuclease which cleaves the DNA [30–33]. For its activity, the FokI domain needs to dimerized [34]; thus, ZFN pairs are required and, hence, designed to bind to the region of interest in the opposite orientation. This also enhances specificity over a longer target region, with site‐specific cleavage of the DNA occurring at the midpoint of the dimerized ZFN pair [35] (Fig. 1). In early studies of ZFNs, the DNA binding domain, imparting specificity to the ZFN, comprised three zinc fingers recognizing a 9‐bp sequence (an 18‐bp composite sequence when the ZFN pairs dimerize). Later, four zinc fingers were engineered to bind a specific sequence of 24 bp as a dimmer and improve specificity. At present, ZFNs can contain up to six zinc fingers facilitating longer and more unique composite recognition site sequences of up to 36 bp [20, 36, 37], potentially allowing for unique and specific targeting at essentially any locus [35]. Further improvements in the design of ZFNs including moving from the initial use of a conventional endonuclease domain to a high‐fidelity endonuclease domain [20, 36, 37], DNA cleavage only upon ZFN pair heterodimerization to minimize cleavage upon homodimerization of ZFNs [36], and the fusion of ZFNs with destabilization domains to regulate their levels [38] have all improved locus specificity and minimized cell toxicity. In essence, a ZFN‐induced double‐strand break (DSB) in the DNA can be specifically and seemingly safely created at any position in a given genome, making them a powerful tool for genetic research. 1 Open in new tabDownload slide Schematic representation of the induction of a DNA double‐strand break (DSB) at a specific genomic site by a zinc finger nuclease (ZFN) pair. Zinc finger proteins (ZFP) modules are engineered to bind to a specific sequence. Each ZFP is able to bind to a certain 3 bp of DNA. Panel (A) shows a module consisting of four ZFPs, which recognizes a composite site of 24 bp (colored sequence). Panel (B) shows the correct binding of a ZFN pair at distinct locations on opposite strands of DNA in close proximity facilitating the dimerization of the FokI domains. The FokI dimer then cleaves the DNA in between the recognition sites of both ZFNs to create the DSB. Abbreviation: ZFPs, zinc finger proteins. 1 Open in new tabDownload slide Schematic representation of the induction of a DNA double‐strand break (DSB) at a specific genomic site by a zinc finger nuclease (ZFN) pair. Zinc finger proteins (ZFP) modules are engineered to bind to a specific sequence. Each ZFP is able to bind to a certain 3 bp of DNA. Panel (A) shows a module consisting of four ZFPs, which recognizes a composite site of 24 bp (colored sequence). Panel (B) shows the correct binding of a ZFN pair at distinct locations on opposite strands of DNA in close proximity facilitating the dimerization of the FokI domains. The FokI dimer then cleaves the DNA in between the recognition sites of both ZFNs to create the DSB. Abbreviation: ZFPs, zinc finger proteins. ZFN‐Mediated Targeted Gene Editing Once a DNA DSB has been induced by ZFN, it can be repaired by intrinsic repair mechanisms of the cell [39]. The break can be repaired by either the nonhomologous end joining (NHEJ) repair pathway or by homology directed repair (HDR). Repair of DNA DSBs by NHEJ and Its Applications in Various Species The NHEJ repair pathway [40] acts to efficiently ligate the broken ends of the DSB together. It is a more error prone pathway than HDR, as there is the possibility for additions or deletions at the break point, which may result in mutation of the locus [41]. This occasional loss or gain in base pairs (bp) following a ZFN‐mediated DSB can be used to produce gene knockouts if the subsequent transcription or translation is erroneous. The repair of DSBs by NHEJ following ZFN‐targeting (Fig. 2.1) can be variable (1%–50%) [37]; however, this is significantly higher than the efficiency reported for HR in mouse models (one in 103 cells), which has so far been the method of choice for generating knockout models. 2 Open in new tabDownload slide Schematic representations of zinc finger nuclease (ZFN)‐mediated genetic modifications. Following induction of a double‐strand break (DSB), the lesion can be repaired in a number of ways. This allows the modification of a region through utilization of the repair machinery. If desired, the addition of an investigator supplied donor DNA can be used for additive genetic modifications. The donor DNA contains regions homologous to the targeted region, which flanks the DNA to be inserted, to facilitate targeted insertion by homologous recombination (HR). Panel (A) illustrates the potential for gene knockout through mutagenic repair by the nonhomologous end joining repair pathway following a ZFN‐induced DSB. Panel (B) portrays gene knockout via HR‐mediated insertional mutagenesis of a selection cassette. Panel (C) shows gene correction following HR with a donor DNA carrying a mutation free sequence. Panel (D) demonstrates gene‐tagging through HR‐mediated insertion of an eGFP cassette. Abbreviations: eGFP, enhanced green fluorescent protein; PGK‐PURO‐pA, phosphoglycerol kinase puromycin‐pA. 2 Open in new tabDownload slide Schematic representations of zinc finger nuclease (ZFN)‐mediated genetic modifications. Following induction of a double‐strand break (DSB), the lesion can be repaired in a number of ways. This allows the modification of a region through utilization of the repair machinery. If desired, the addition of an investigator supplied donor DNA can be used for additive genetic modifications. The donor DNA contains regions homologous to the targeted region, which flanks the DNA to be inserted, to facilitate targeted insertion by homologous recombination (HR). Panel (A) illustrates the potential for gene knockout through mutagenic repair by the nonhomologous end joining repair pathway following a ZFN‐induced DSB. Panel (B) portrays gene knockout via HR‐mediated insertional mutagenesis of a selection cassette. Panel (C) shows gene correction following HR with a donor DNA carrying a mutation free sequence. Panel (D) demonstrates gene‐tagging through HR‐mediated insertion of an eGFP cassette. Abbreviations: eGFP, enhanced green fluorescent protein; PGK‐PURO‐pA, phosphoglycerol kinase puromycin‐pA. The first targeting of a locus using designed ZFNs followed by NHEJ‐mediated repair was carried out in Drosophila melanogaster on the yellow gene [32]. To date, this type of ZFN‐mediated gene disruption has been carried out in a diverse variety of model organisms, from plants to mammals, demonstrating the broad application of ZFNs across different cell types in many species [42–51]. Recent work has revealed that the sequential or simultaneous application of multiple ZFNs is possible to engineer double gene knockouts at distinct loci in Chinese hamster ovary (CHO) cells [52] and even triple gene knockouts in the transformed human K562 cell line [53]. Despite a few exceptions [54, 55], where classical gene targeting was used to disrupt a gene of interest, RNAi has predominantly been used to knockdown gene expression to help elucidate gene function in human cells [56]. However, it is now possible to achieve specific knockout of a gene in human cells through ZFN NHEJ‐mediated disruption of a target locus without selection in a significant proportion of the experimental cell population. Repair of DNA DSBs by HDR DSBs are recombinogenic and can thus be repaired by the HDR pathway [57]. In this case, a DNA DSB is repaired using the homologous sequence from the undamaged sister chromatid as a template. It has been shown that the HDR apparatus can use a supplied donor DNA plasmid that contains homology arms as a surrogate template [7, 58]. This approach allows for gene correction of single nucleotide changes from an exogenous episomal donor to the endogenous locus (Fig. 2.2). Thus, point mutations can be created and corrected to study and potentially cure genetic diseases. ZFNs used in combination with a DNA donor template have been shown to increase gene editing efficiency, initially in drosophila [31], and, for the first time in mammalian cells, in human transformed HEK293 cells [59]. The correction and creation of point mutations in the interleukin‐2 receptor‐γ (IL2RG) gene in the human transformed cell line K562 and human CD4+ T‐cells was subsequently demonstrated with 20% of clones acquiring the donor specified correction at this site, 8% of these showing modification at both alleles in K562 cells [35]. Larger sections of DNA can also be inserted into the genome at a desired location using this technique. ZFNs directed to IL2RG in human cells were used to demonstrate this in combination with episomal donors carrying 750 bp homology arms (to the chromosomal DNA either side of the ZFN target site), which flank a nonhomologous transgene. In this case, 5% of chromatids gained donor transgenes of up to 8 kb in length, without selection [58]. This methodology has been used to create a novel allelic form of a gene and an isogenic panel of cell lines, for example, mouse ESC lines of defined series of alleles for an endogenous gene [60], and to tag and add transgenes to an endogenous locus in human cells [19, 20, 22, 58]. Therefore, in comparison to classical gene targeting, ZFNs have been shown to increase the rate of HDR at a specific site in the genome in human cells [19‐22, 35, 37, 59, 61] and thereby efficiently create site‐specific genome modifications in cell lines and model organisms (Fig. 2.1–2.4). ZFNS and hPSCS The first published example of ZFN‐mediated genetic manipulation in hESCs used the infectivity of a lentiviral vector to deliver the ZFN cassette and donor construct into hESC [22]. An integration‐defective lentiviral vector was used to minimize the toxicity associated with random genomic integration [62–66]. Gene editing in hESCs was carried out on the chemokine (C‐C motif) receptor 5 (CCR5) gene as the homozygous null mutation of this gene appears to be well tolerated in human cells [67]. ZFN‐mediated addition of a green fluorescent protein (GFP) expression cassette at the CCR5 locus, without positive selection, was successful in 5% of hESCs (Table 1). The successfully targeted cells showed stable transgene expression, maintained the pluripotent phenotype, and the ability to differentiate into neural progenitors with the continued expression of GFP. Analysis of hESCs treated with the ZFNs showed that 28% of the CCR5 alleles had mutations at the ZFN target site indicating NHEJ repair of the DSB and the ability to disrupt a gene using ZFNs in hESCs [22]. Table 1 Summary of zinc finger nuclease‐driven endogenous gene targeting reported in human pluripotent stem cells Open in new tab Table 1 Summary of zinc finger nuclease‐driven endogenous gene targeting reported in human pluripotent stem cells Open in new tab The potential and limitations of this pioneering study by Lombardo et al. [22] have been discussed by others [1, 21]. A number of these limitations including, lack of confirmation for the targeted HDR event and presence of any random integrations of the donor DNA, use of hESC lines with a stable karyotype during prolonged culture, and the use of a viral vector have been addressed in subsequent publications [19–21]. The findings of these studies are outlined below in five key applications of ZFNs in hPSCs to date and a summary of the procedures achieved and methodology used is shown in Tables 1 and 2. Table 2 Summary of zinc finger nuclease‐driven targeting to the AAVS1 locus in human pluripotent stem cells Open in new tab Table 2 Summary of zinc finger nuclease‐driven targeting to the AAVS1 locus in human pluripotent stem cells Open in new tab Genetic Modification for Correction or Disruption of the Desired Gene Correction of a defective enhanced GFP (eGFP) inserted at a chromosomal locus by Zou et al. [21] demonstrated the possibility for specific genetic alterations at an endogenous location (for curative purposes, through mutation correction, or creating a desired mutation for disease modeling in an appropriate human cell type). Zou et al. [21] also demonstrated the ability to knockout gene function. This was possible either through mutagenic NHEJ repair with addition of ZFNs alone or in combination with a donor DNA for insertional mutagenesis of the disease‐related phosphatidylinositol glycan anchor biosynthesis, class A (PIG‐A) gene. The PIG‐A gene is mutated in hematopoietic stem cells from patients with the blood disorder paroxysmal nocturnal hemoglobinuria. Both of these approaches were successfully carried out in hESC and human induced pluripotent stem cells (hiPSCs) and have implications for therapies or gene function studies. Introduction of Reporter Genes into Endogenous Loci to Enable Monitoring of Pluripotency or Tracking of Cellular Differentiation Hockemeyer et al. demonstrated the ability to monitor maintenance of pluripotency by successfully tagging eGFP reporter into the octamer‐binding protein 4 (POU5F1, POU class 5 homeobox 1) (OCT4) locus of hESCs [20]. OCT4 is one of the few genes that has previously been successfully targeted in hESCs using a conventional HR method [14]. The inclusion of a puromycin resistance cassette allows for selection of clones where integration has occurred. This resulted in an enhanced targeting efficiency with more than 94% of puromycin resistant clones being correctly targeted. Introduction of eGFP reporter did not affect the pluripotent phenotype as the OCT4‐eGFP hESCs expressed pluripotency markers and were able to differentiate into cell types from all three developmental germ layers [20]. Additionally, Hockemeyer et al. [20] showed that further reporting on the fate of a stem cell can be achieved as it differentiates. They are able to target a transcriptionally inactive gene, paired‐like homeodomain transcription factor 3 (PITX3), in hPSCs with specific ZFNs to create an eGFP‐PITX3 fusion at the endogenous locus. PITX3 is a transcription factor that is not expressed in hPSCs but in differentiated cells. Correct targeting was achieved with 11% efficiency in hESCs and 8% efficiency in two hiPSC lines, with all lines retaining a normal karyotype [20]. The ability to efficiently target even transcriptionally inactive genes in undifferentiated stem cells will be useful for the reporting on expression of specific genes and the emergence of specific cell types during differentiation to improve understanding and protocols for differentiation of hPSCs to various cell types. Targeting of Expression Cassettes into the AAVS1 “Safe Harbor” Locus for Their Constitutive or Inducible Expression ZFNs have been designed and used to target the AAVS1 locus [19, 20], which encodes the ubiquitously expressed protein phosphatase 1, regulatory subunit 12C (PPP1R12C) gene. This locus is commonly targeted for long‐term stable transgene expression in a number of cell types including hESCs [68]. Integration of adeno‐associated virus (AAV) disrupts the PPP1R12C gene on chromosome 19, commonly referred to as the AAVS1 locus [69, 70]; however, this targeting event is not associated with any pathophysiology [68]. Both hESCs [19, 20, 68] and hiPSCs [20] with a disrupted PPP1R12C gene, either through conventional gene targeting [68] or ZFN‐driven gene addition [19, 20], proliferate normally, have a normal karyotype, express pluripotency markers, and maintain pluripotency. The PPP1R12C promoter is active in hPSCs driving sufficient expression of transgene to facilitate selection of pools and clones of cells with correctly targeted promoterless markers [19, 20]. Thus, the AAVS1 locus is designated a safe harbor as there are no deleterious effects following its disruption and stable and prolonged expression of the transgene in many cell types with both promoterless and promoter‐containing cassettes. In addition, delivery to a safe harbor can achieve isogenic settings for analysis due to the specificity of ZFN‐mediated gene addition at this site and hence the avoidance of random integrations in hPSCs. Other methods of transgene addition into the AAVS1 locus in human cells exist, such as a two‐step recombination‐based cassette exchange [71] and gene targeting with recombinant AAV vectors [72]; however, the ZFN approach complements and increases the tools for targeted delivery to this locus. Two studies, from Hockemeyer et al. [20] and Dekelver et al. [19] demonstrated that a number of different expression cassettes can be targeted to and integrated into the AAVS1 locus in hESCs and hiPSCs in a single step using plasmid DNA constructs (see Table 2 for summary). Robust and long‐term expression of the transgene can be achieved either with the native PPP1R12C promoter or exogenous promoters in the inserted expression cassette, with no effect on cell growth. Also expression cassettes driven by the exogenous promoters such as phosphoglycerol kinase, cytomegalovirus (CMV), and CMV‐enhancer/chicken β‐actin (CAGGS) were successfully targeted to AAVS1 [19, 20]. It is also possible to achieve inducible expression of cassettes at this locus. This has been shown with tetracycline response element driven expression cassettes in hESCs and hiPSCs with a dose‐dependent control of expression following transduction with a lentivirus carrying M2rtTA (transactivator for cells responsive to doxycycline) and addition of doxycycline [20]. Furthermore, DeKelver et al. [19] show that it is possible to create, in one single step, a hESC transheterozygous at the AAVS1 locus for four separate genetic entities, which together comprise an inducible gene expression system. They use the fact that the AAVS1 locus is autosomal; thus, a euploid cell has two distinct genetic locations for transgenes at each allele. Both alleles were targeted by using two different selectable markers to isolate transheterozygotes. Simultaneous treatment of hESCs with AAVS1 ZFNs and two different donor DNAs, each with a promoterless distinct selectable marker followed by an expression cassette for either the constitutive expression of the tetracycline reverse transactivator M2rtTA or inducible expression of a histone H2B‐eGFP fusion was used to generate the transheterozygotes. Genotyping of single cell derived clones resistant to both selection agents showed that they were transheterozygotes for both donor cassettes at the AAVS1 locus. H2B‐eGFP fusion protein showing condensed chromosomes in mitosis was shown following doxycycline treatment. Thus, the AAVS1 locus was able to facilitate the long‐term expression and function of four separate expression cassettes, two promoterless resistance markers (driven by the PPP1R12C promoter), and two separate promoter‐transcription units without affecting cell division or morphology. Knockdown of Gene Expression Through Targeted Insertion of Short Hairpin RNAs (shRNAs) to a “Safe Harbor” Locus DeKelver et al. [19] demonstrated the feasibility of targeting shRNA cassettes to the AAVS1 locus in hESC. Over recent years, RNAi technology has enabled loss of gene function studies [56, 73, 74]. The use of shRNA cassettes has been through transient expression or random integration with lentivirus transgenesis of the shRNA cassette. Authors show that shRNAs against tumor protein p53, DNA‐methyltransferase 1, or control shRNA expression cassette donor constructs and AAVS1 specific ZFNs were used to generate single‐cell derived hESC clones. Clones with target‐specific shRNA cassettes, but not the control shRNA, showed stable long‐term knockdown of target mRNA levels [19]. Therefore, it is possible to rapidly produce single cell derived clones, without aberrant insertions, isogenic save for a shRNA cassette against a gene of choice, and with long‐term robust gene knockdown. Additional Uses for ZFNs as Tools for Studies in hPSC Biology Brunet et al. [18] used ZFNs to induce DSBs at specific loci in hESCs to study chromosomal translocations in human cells and analyze the impact of these translocations, which are hallmarks of several tumor types. Recurrent chromosomal translocations associated with cancers are considered to be an initiating event in tumorigenesis and in some cases are thought to occur in stem and progenitor cells [75, 76]. Therefore, the high prevalence of translocations in human malignancy warrants further investigation regarding mechanisms and effects, particularly in relevant primary human cell types, such as stem and progenitor cells, where it has been lacking. This article presents an approach to induce translocations in human cells at specified loci. Concurrent DNA DSBs were induced at two endogenous loci, the PPP1R12C gene on chromosome 19, and the IL2Rγ gene on the X chromosome in hESCs. Translocations, detected by nested quantitative polymerase chain reaction (PCR) of translocation breakpoint junctions for t (19;X), were detected in hESCs. This ZFN approach is efficient and applicable to primary cells, such as hESCs, because of the least genetic manipulation and short culture afforded by ZFNs, thereby, allowing a rapid and efficient induction of site‐specific DSBs for the study of the mechanisms and phenotypes associated with specific targeted translocations [18]. These applications demonstrate the usefulness and extent of successful genetic modifications with ZFNs. It is important to note that most of these have been achieved with the use of nonviral vectors (Tables 1 and 2) and delivery methods, such as electroporation [19, 20] and nucleofection [18, 21]. This is beneficial for the future clinical applications of stem cell therapies. However, the nonviral delivery systems are less efficient than their viral counterparts in hPSCs, and therefore, further developments of various delivery systems for stem cells such as the use of nanoparticles [77] are eagerly awaited. The first results from a phase I safety trials using ZFN were recently presented at the Conference on Retroviruses and Opportunistic infections by Sangamo Biosciences. This trial involved ZFN targeting of CD34+ cells isolated from male patients with HIV who were treated with conventional antiviral therapies followed by reinfusion. It is know that homozygous deletion of CCR5 confer resistance to HIV infections, and for this reason, ZFNs were designed to target and disrupt the CCR5 gene. The results of this trial showed promising results with a boost in numbers of immune cells in five of six patients and no significant side effects. Although the efficiency and efficacy of this treatment still remain to be evaluated, this clinical application does reflect the potential of ZFNs in targeting and genetically manipulating the stem cell compartment. Potential Problems Associated with ZFN‐Mediated Genomic Editing ZFN Specificity Key to ZFN‐mediated genetic targeting is the specificity of the ZFNs used. This is highly important not only to enable targeted cutting at the desired site but also to prevent any off‐target cutting, which can lead to reduced cutting efficiency at the target site, misleading experimental interpretation, and increase toxicity to the cell due to possible deleterious side effects. For example, DSBs have been shown to be a source of oncogenic translocations [78, 79] and ZFNs through the induction of DSBs have been used to induce translocations [18]. These are rare events, but due to their potential danger, it is important to detect whether ZFNs are creating such translocations. A number of factors inherent in the design of a ZFN have led to the production of highly specific targeting nucleases and reduced the potential for off‐target cleavage. Primarily, the interaction of the ZFPs of a designed ZFN at the correct positions across the entire recognition interface of the DNA target site [35, 37, 43, 45]. This can be achieved and enhanced by changing residues or entire α‐helices in a ZFP for improved binding in vivo, based on information from previously used ZFP modules [35]. Furthermore, multiple two‐finger modules have been used to increase the recognition site to 12–18 bp. These properties result in a unique or rare specific binding site in the genome for the ZFN. In addition, the use of obligate heterodimerization domains to promote formation of a heterodimer of particular ZFN pairs for induction of a DSB [36, 80] and the ability to control the timing and extent of ZFN expression by using small molecules [38] minimizes the chance of off‐site targeting by the ZFNs. A number of studies have shown that hESCs and iPSCs following ZFN‐mediated targeting display no karyotypic abnormalities or any changes in pluripotency after prolonged culture [19–21], which would suggest minimal toxicity with the ZFNs used. Although, major genomic alteration could lead to karyotypic or proliferative abnormalities, or cell death; minor alterations (small additions or deletions), however, could be maintained in the population and may only become apparent or deleterious over time. ZFNs must also be used at an appropriate concentration as ZFN‐mediated toxicity was observed when used at a high concentration [21]. In addition, the high specificity of ZFNs and ZFN‐mediated gene addition can be indicated by a nucleus‐wide DSB‐frequency and the rate of random integration of donor plasmid [19, 59]. Testing Integrations Accuracy of the integration of donor DNA should be tested to ascertain presence of correct integration, clonality of the isolated clones, and any additional integration. Southern blot analysis with internal and external probes to the inserted region is used in a number of reports [19–21] to determine whether there are any additional integrations and the homogeneity of the targeted clones. Confirmation of the presence of the donor DNA can be revealed by PCR with primers that anneal outside the region of homology and to the inserted donor [19]. To check whether donor DNA addition to the target site occurred solely in a homology‐dependant process, the transgenic region can be sequenced [19] as there is the possibility that transgene addition could occur through a combination of HDR and NHEJ repair mechanisms [79, 81]. Testing for Off‐Target Cleavage Despite the advances in ZFN technology and reports on the rarity of off‐site targeting, off‐target cleavage by ZFNs can occur. ZFN infidelity has in fact been observed in hESCs, in a screen for mutation at the most probable off‐target sites for the ZFNs used. Therefore, screening of clones at the most likely off‐target cutting sites, or of the whole genome, needs to be carried out to maintain an isogenic setting and for any future therapeutic uses. The systematic evolution of ligands by exponential enrichment (SELEX) has been used to experimentally determine the consensus binding site of a ZFP [82, 83]. SELEX identifies nucleic acid target sequence of a protein molecule, involving the incubation of the protein molecule (bait) with a randomized library of nucleic acid ligands. The bait protein is subsequently isolated with any bound nucleic acids, which are then identified by PCR and sequencing. This has been done for naturally occurring ZFPs [84] and this information has been used to perform a bioinformatic study of the genome of interest. Regions of high similarity to the consensus site and thus potential off‐target sites can then be found and interrogated by sequencing these sites to look for any changes follow ZFN targeting. This has shown no off‐target editing by ZFNs, in a number of systems, including zebrafish [43], rats [45], maize [85], and human T‐cells [37], save for one exception in hESCs [20]. A screen of the 10 most probable off‐target sites for mutations from four different ZFN‐mediated targeting experiments in hESCs was performed. The potential off‐target sites tested were wild‐type except for a single site in one of four (1/4) clones correctly targeted for the PITX3 gene, which carried an off‐target NHEJ mutation [20]. Further indirect approaches have been used to assess the genome integrity following ZFN activity, for instance, immunostaining for markers of DSBs such as γH2AX and p53‐binding protein [36]. A single method of analysis may not be sufficient to fully and accurately assess specificity of ZFNs and hence multiple methods have been used. However, a more sensitive method to measure off‐target affects of ZFNs and assess any genetic editing is high‐throughput sequencing. This was used to assess the ZFN targeting of the CCR5 gene and showed that the ZFNs used were highly specific for the CCR5 gene with the closet paralogue, the CCR2 gene, showing around a 10 fold less likely preference for the ZFNs. Of interest, however, was the occurrence of very rare off‐target events found with a one in 20,000 occurrence in the intron of the actin binding LIM protein family, member 2 gene [37], indicating that for complete assurance for the absence of any off‐target affects high‐throughput sequencing of a significant number of targeted clones is necessary to pick up rare and unexpected ZFN cleavage. In the future, as high‐throughput sequencing technology becomes more viable and affordable, screening of clones may allow isolation of the ideal clones for experimentation or therapeutics. Application of high concentrations of a particular ZFN pair could also be carried out to indicate any potential common off‐target sites. This information could then be used in subsequent experiments to test these sites for mutations. Further Problems Associated with ZFN‐Driven Addition of Genetic Material to a Desired Targeted Locus These include combined delivery of ZFN template and donor DNA into the cell with the aim of maximizing efficiency whilst minimizing toxicity. Viral [22] and nonviral [18–21] delivery systems have been successfully used in hPSCs (Tables 1 and 2). Both systems exhibit sufficient efficiency to allow successful targeting. The added safety assurance of the nonviral systems is an additional benefit of ZFN‐mediated targeting and makes these the most attractive option for future therapies. In addition, the pathway used to repair the ZFN‐induced DSB is able to cause potential problems. For example, the preferential use of NHEJ repair instead of HDR would lower gene addition efficiency at the targeted locus and potentially lead to mutagenesis. It is of interest to note that in some of the heterozygously targeted clones tested the nonedited allele has been targeted but repaired by NHEJ [20]. In the Hockemeyer et al. [20] study, one of 11 clones successfully targeted with the OCT4‐ZFNs and one of 18 with the PITX3‐ZFNs had NHEJ repair‐mediated mutations at the second nonedited allele of the targeted locus, rather than HR with the donor DNA (or undamaged sister chromatid). At present, the preference for one DSB repair mechanism over the other has not been fully elucidated. However, it is possible to target intronic regions with ZFNs to ensure that a wild‐type allele is present in heterozygously targeted clones. Recently, it has been shown that the absence of ligase 4 activity in drosophila pushes the repair mechanisms strongly toward HDR [86]. Whether this is the case in human cells remains to be determined. Once discovered, this could enable repair solely with one mechanism or the other to avoid incurring potentially deleterious mutations with NHEJ when HDR is desired for gene correction or addition. Therapeutic Applications for ZFNS in hPSCS The applications of ZFNs in hPSCs has great potential for efficient gene targeting and genetic engineering at a desired endogenous location (Table 3). These modifications, such as gene targeting, have therapeutic implications. The main therapeutic application touted for ZFNs in hPSCs is reliant on the recent ability to reprogram human somatic cells into iPSCs [87–91], which display the similar stemness characteristics of hESCs. As a result, this has fueled great hope for patient specific therapies with personalized cell replacement. The correction of inherited mutations in patient stem cells has been a hope for curative regenerative medicine [92] and thus the specific and permanent ZFN‐mediated correction of an endogenous disease causing mutation in patient derived hiPSCs, followed by differentiation of the correctly targeted hiPSC clone to a specific cell type for transplantation and repopulation of the desired affected location, is an attractive proposition. A targeted approach for such gene corrections is paramount not only for the endogenous genetic control and environment but also to prevent possible random integrations [93–96]. This is only reinforced by the use of gene therapy to cure severe combined immunodeficiency patients, where activation of an oncogene due to integration caused transformed cell growth and the patients developed leukemia [97]. Table 3 Applications and therapeutic potential of zinc finger nuclease‐driven genetic alterations in human pluripotent stem cells Open in new tab Table 3 Applications and therapeutic potential of zinc finger nuclease‐driven genetic alterations in human pluripotent stem cells Open in new tab Successful gene targeting in hiPSCs has been shown [20, 21], demonstrating feasibility of ZFN gene targeting applications in research and therapy for disease‐specific hiPSCs. Thus, an efficient system exists to allow the correction of disease‐causing mutations, including serial gene targeting to create specific and biallelic modifications. These abilities alongside studies of successful iPSC generation and differentiation, and the correction of disease‐causing mutations in patient‐specific iPSCs and subsequent use of the genetically corrected cells to cure a disease in mice [98] brings patient‐specific regenerative therapies closer. Conversely, the creation of disease‐specific mutations using ZFNs in hPSCs for human cell models of various human diseases, particularly developmental conditions where differentiation down a certain lineage could indicate the role of a mutation in a particular developmental defect, could allow the study of disease and drug therapies in relevant cell types. This strategy is of particular importance for diseases such as Fanconi anemia, where approaches to create a hiPSCs model have been unsuccessful as correction of the defect has been required for reprogramming of affected patient cells [99]. Conclusion ZFN technology is still in its relative infancy; however, at present, application of ZFNs has great potential for experimental work and future clinical applications. The ability to specifically and efficiently target and edit a locus of interest is highly prized and ZFNs readily facilitate this. The use of ZFNs in hPSCs is already proving successful and the ability to efficiently target a genetic locus in primary cells makes this technology even more enticing. ZFN‐driven genetic editing has been shown to allow complete gene knockout, constitutive and inducible expression of a transgene, gene tagging, use of RNAi technology, and use of a safe harbor locus, all with minimal effect on other loci but the targeted one. Further study and continued successful applications of this technology will only strengthen the potential to achieve specific genetic editions in an isogenic background to provide clear insight and curative effects. However, with every new technology, there are inherent risks and these need to be understood and prevented for the application of ZFNs to reach the potential heights proposed, much work has been done on this already and has been sufficient to begin clinical trials with ZFN‐driven correction of human cells. Nevertheless, unwanted alterations have been detected in hESCs and enough needs to be done to ensure safe application of ZFNs. The marriage of this and other new technologies such as high‐throughput sequencing could allow complete determination of successful targeting and the absence of other alterations. This particularly needs to be the case for therapy with ZFN‐mediated gene targeting as unforeseen deleterious genetic alterations can be devastating. However, the ability of hPSCs to self‐renew may negate the need for perfection in every successfully edited clone derived as each clone can be studied at length to ascertain true successful targeting without any additional genetic alterations. With patient‐derived hiPSCs, the promise of gene correction in monogenic disease and even more complicated alterations in complex diseases in an allogenic setting may well be achievable. Disclosure of Potential Conflicts of Interest The authors declare they have no potential conflicts of interests. REFERENCES 1 Giudice A , Trounson A. Genetic modification of human embryonic stem cells for derivation of target cells . Cell Stem Cell 2008 ; 2 : 422 – 433 . Google Scholar Crossref Search ADS PubMed WorldCat 2 Stewart R , Yang C, Anyfantis G et al. Silencing of the expression of pluripotent driven‐reporter genes stably transfected into human pluripotent cells . Regen Med 2008 ; 3 : 505 – 522 . Google Scholar Crossref Search ADS PubMed WorldCat 3 Thomas KR , Folger KR, Capecchi MR. High frequency targeting of genes to specific sites in the mammalian genome . Cell 1986 ; 44 : 419 – 428 . Google Scholar Crossref Search ADS PubMed WorldCat 4 Capecchi MR . Generating mice with targeted mutations . Nat Med 2001 ; 7 : 1086 – 1090 . Google Scholar Crossref Search ADS PubMed WorldCat 5 Evans MJ . The cultural mouse . Nat Med 2001 ; 7 : 1081 – 1083 . Google Scholar Crossref Search ADS PubMed WorldCat 6 Smithies O . Forty years with homologous recombination . Nat Med 2001 ; 7 : 1083 – 1086 . Google Scholar Crossref Search ADS PubMed WorldCat 7 Thomson JA , Itskovitz‐Eldor J, Shapiro SS et al. Embryonic stem cell lines derived from human blastocysts . Science 1998 ; 282 : 1145 – 1147 . Google Scholar Crossref Search ADS PubMed WorldCat 8 Costa M , Dottori M, Sourris K et al. A method for genetic modification of human embryonic stem cells using electroporation . Nat Protoc 2007 ; 2 : 792 – 796 . Google Scholar Crossref Search ADS PubMed WorldCat 9 Davis RP , Ng ES, Costa M et al. Targeting a GFP reporter gene to the MIXL1 locus of human embryonic stem cells identifies human primitive streak‐like cells and enables isolation of primitive hematopoietic precursors . Blood 2008 ; 111 : 1876 – 1884 . Google Scholar Crossref Search ADS PubMed WorldCat 10 Di Domenico AI , Christodoulou I, Pells SC et al. Sequential genetic modification of the hprt locus in human ESCs combining gene targeting and recombinase‐mediated cassette exchange . Cloning Stem Cells 2008 ; 10 : 217 – 230 . Google Scholar Crossref Search ADS PubMed WorldCat 11 Irion S , Luche H, Gadue P et al. Identification and targeting of the ROSA26 locus in human embryonic stem cells . Nat Biotechnol 2007 ; 25 : 1477 – 1482 . Google Scholar Crossref Search ADS PubMed WorldCat 12 Ruby KM , Zheng B. Gene targeting in a HUES line of human embryonic stem cells via electroporation . Stem Cells 2009 ; 27 : 1496 – 1506 . Google Scholar Crossref Search ADS PubMed WorldCat 13 Urbach A , Schuldiner M, Benvenisty N. Modeling for Lesch‐Nyhan disease by gene targeting in human embryonic stem cells . Stem Cells 2004 ; 22 : 635 – 641 . Google Scholar Crossref Search ADS PubMed WorldCat 14 Zwaka TP , Thomson JA. Homologous recombination in human embryonic stem cells . Nat Biotechnol 2003 ; 21 : 319 – 321 . Google Scholar Crossref Search ADS PubMed WorldCat 15 Carroll D . Progress and prospects: zinc‐finger nucleases as gene therapy agents . Gene Ther 2008 ; 15 : 1463 – 1468 . Google Scholar Crossref Search ADS PubMed WorldCat 16 Klug A . The discovery of zinc fingers and their applications in gene regulation and genome manipulation . Annu Rev Biochem 2010 ; 79 : 213 – 231 . Google Scholar Crossref Search ADS PubMed WorldCat 17 Urnov FD , Rebar EJ, Holmes MC et al. Genome editing with engineered zinc finger nucleases . Nat Rev Genet 2010 ; 11 : 636 – 646 . Google Scholar Crossref Search ADS PubMed WorldCat 18 Brunet E , Simsek D, Tomishima M et al. Chromosomal translocations induced at specified loci in human stem cells . Proc Natl Acad Sci USA 2009 ; 106 : 10620 – 10625 . Google Scholar Crossref Search ADS PubMed WorldCat 19 DeKelver RC , Choi VM, Moehle EA et al. Functional genomics, proteomics, and regulatory DNA analysis in isogenic settings using zinc finger nuclease‐driven transgenesis into a safe harbor locus in the human genome . Genome Res 2010 ; 20 : 1133 – 1142 . Google Scholar Crossref Search ADS PubMed WorldCat 20 Hockemeyer D , Soldner F, Beard C et al. Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc‐finger nucleases . Nat Biotechnol 2009 ; 27 : 851 – 857 . Google Scholar Crossref Search ADS PubMed WorldCat 21 Zou J , Maeder ML, Mali P et al. Gene targeting of a disease‐related gene in human induced pluripotent stem and embryonic stem cells . Cell Stem Cell 2009 ; 5 : 97 – 110 . Google Scholar Crossref Search ADS PubMed WorldCat 22 Lombardo A , Genovese P, Beausejour CM et al. Gene editing in human stem cells using zinc finger nucleases and integrase‐defective lentiviral vector delivery . Nat Biotechnol 2007 ; 25 : 1298 – 1306 . Google Scholar Crossref Search ADS PubMed WorldCat 23 Durai S , Mani M, Kandavelou K et al. Zinc finger nucleases: Custom‐designed molecular scissors for genome engineering of plant and mammalian cells . Nucleic Acids Res 2005 ; 33 : 5978 – 5990 . Google Scholar Crossref Search ADS PubMed WorldCat 24 Porteus MH , Carroll D. Gene targeting using zinc finger nucleases . Nat Biotechnol 2005 ; 23 : 967 – 973 . Google Scholar Crossref Search ADS PubMed WorldCat 25 Miller J , McLachlan AD, Klug A. Repetitive zinc‐binding domains in the protein transcription factor IIIA from Xenopus oocytes . EMBO J 1985 ; 4 : 1609 – 1614 . Google Scholar Crossref Search ADS PubMed WorldCat 26 Choo Y , Klug A. Toward a code for the interactions of zinc fingers with DNA: selection of randomized fingers displayed on phage . Proc Natl Acad Sci USA 1994 ; 91 : 11163 – 11167 . Google Scholar Crossref Search ADS PubMed WorldCat 27 Pabo CO , Peisach E, Grant RA. Design and selection of novel Cys2His2 zinc finger proteins . Annu Rev Biochem 2001 ; 70 : 313 – 340 . Google Scholar Crossref Search ADS PubMed WorldCat 28 Rebar EJ , Pabo CO. Zinc finger phage: Affinity selection of fingers with new DNA‐binding specificities . Science 1994 ; 263 : 671 – 673 . Google Scholar Crossref Search ADS PubMed WorldCat 29 Tan S , Guschin D, Davalos A et al. Zinc‐finger protein‐targeted gene regulation: Genomewide single‐gene specificity . Proc Natl Acad Sci USA 2003 ; 100 : 11997 – 12002 . Google Scholar Crossref Search ADS PubMed WorldCat 30 Kim YG , Cha J, Chandrasegaran S. Hybrid restriction enzymes: Zinc finger fusions to Fok I cleavage domain . Proc Natl Acad Sci USA 1996 ; 93 : 1156 – 1160 . Google Scholar Crossref Search ADS PubMed WorldCat 31 Bibikova M , Carroll D, Segal DJ et al. Stimulation of homologous recombination through targeted cleavage by chimeric nucleases . Mol Cell Biol 2001 ; 21 : 289 – 297 . Google Scholar Crossref Search ADS PubMed WorldCat 32 Bibikova M , Golic M, Golic KG et al. Targeted chromosomal cleavage and mutagenesis in Drosophila using zinc‐finger nucleases . Genetics 2002 ; 161 : 1169 – 1175 . Google Scholar Crossref Search ADS PubMed WorldCat 33 Bibikova M , Beumer K, Trautman JK et al. Enhancing gene targeting with designed zinc finger nucleases . Science 2003 ; 300 : 764 . Google Scholar Crossref Search ADS PubMed WorldCat 34 Vanamee ES , Santagata S, Aggarwal AK. FokI requires two specific DNA sites for cleavage . J Mol Biol 2001 ; 309 : 69 – 78 . Google Scholar Crossref Search ADS PubMed WorldCat 35 Urnov FD , Miller JC, Lee YL et al. Highly efficient endogenous human gene correction using designed zinc‐finger nucleases . Nature 2005 ; 435 : 646 – 651 . Google Scholar Crossref Search ADS PubMed WorldCat 36 Miller JC , Holmes MC, Wang J et al. An improved zinc‐finger nuclease architecture for highly specific genome editing . Nat Biotechnol 2007 ; 25 : 778 – 785 . Google Scholar Crossref Search ADS PubMed WorldCat 37 Perez EE , Wang J, Miller JC et al. Establishment of HIV‐1 resistance in CD4+ T cells by genome editing using zinc‐finger nucleases . Nat Biotechnol 2008 ; 26 : 808 – 816 . Google Scholar Crossref Search ADS PubMed WorldCat 38 Pruett‐Miller SM , Reading DW, Porter SN et al. Attenuation of zinc finger nuclease toxicity by small‐molecule regulation of protein levels . PLoS Genet. 2009 ; 5 : e1000376 . Google Scholar Crossref Search ADS PubMed WorldCat 39 Jackson SP , Bartek J. The DNA‐damage response in human biology and disease . Nature 2009 ; 461 : 1071 – 1078 . Google Scholar Crossref Search ADS PubMed WorldCat 40 Lieber MR . The mechanism of double‐strand DNA break repair by the nonhomologous DNA end‐joining pathway . Annu Rev Biochem 2010 ; 79 : 181 – 211 . Google Scholar Crossref Search ADS PubMed WorldCat 41 Lieber MR . The mechanism of human nonhomologous DNA end joining . J Biol Chem 2008 ; 283 : 1 – 5 . Google Scholar Crossref Search ADS PubMed WorldCat 42 Beumer KJ , Trautman JK, Bozas A et al. Efficient gene targeting in Drosophila by direct embryo injection with zinc‐finger nucleases . Proc Natl Acad Sci USA 2008 ; 105 : 19821 – 19826 . Google Scholar Crossref Search ADS PubMed WorldCat 43 Doyon Y , McCammon JM, Miller JC et al. Heritable targeted gene disruption in zebrafish using designed zinc‐finger nucleases . Nat Biotechnol 2008 ; 26 : 702 – 708 . Google Scholar Crossref Search ADS PubMed WorldCat 44 Foley JE , Yeh JR, Maeder ML et al. Rapid mutation of endogenous zebrafish genes using zinc finger nucleases made by Oligomerized Pool ENgineering (OPEN) . Plos One 2009 ; 4 : e4348 . Google Scholar Crossref Search ADS PubMed WorldCat 45 Geurts AM , Cost GJ, Freyvert Y et al. Knockout rats via embryo microinjection of zinc‐finger nucleases . Science 2009 ; 325 : 433 . Google Scholar Crossref Search ADS PubMed WorldCat 46 Lloyd A , Plaisier CL, Carroll D et al. Targeted mutagenesis using zinc‐finger nucleases in Arabidopsis . Proc Natl Acad Sci USA 2005 ; 102 : 2232 – 2237 . Google Scholar Crossref Search ADS PubMed WorldCat 47 Mashimo T , Takizawa A, Voigt B et al. Generation of knockout rats with X‐linked severe combined immunodeficiency (X‐SCID) using zinc‐finger nucleases . Plos One 2010 ; 5 : e8870 . Google Scholar Crossref Search ADS PubMed WorldCat 48 Meng X , Noyes MB, Zhu LJ et al. Targeted gene inactivation in zebrafish using engineered zinc‐finger nucleases . Nat Biotechnol 2008 ; 26 : 695 – 701 . Google Scholar Crossref Search ADS PubMed WorldCat 49 Osakabe K , Osakabe Y, Toki S. Site‐directed mutagenesis in Arabidopsis using custom‐designed zinc finger nucleases . Proc Natl Acad Sci USA 2010 ; 107 : 12034 – 12039 . Google Scholar Crossref Search ADS PubMed WorldCat 50 Siekmann AF , Standley C, Fogarty KE et al. Chemokine signaling guides regional patterning of the first embryonic artery . Genes Dev 2009 ; 23 : 2272 – 2277 . Google Scholar Crossref Search ADS PubMed WorldCat 51 Zhang F , Maeder ML, Unger‐Wallace E et al. High frequency targeted mutagenesis in Arabidopsis thaliana using zinc finger nucleases . Proc Natl Acad Sci USA 2010 ; 107 : 12028 – 12033 . Google Scholar Crossref Search ADS PubMed WorldCat 52 Cost GJ , Freyvert Y, Vafiadis A et al. BAK and BAX deletion using zinc‐finger nucleases yields apoptosis‐resistant CHO cells . Biotechnol Bioeng 2010 ; 105 : 330 – 340 . Google Scholar Crossref Search ADS PubMed WorldCat 53 Liu PQ , Chan EM, Cost GJ et al. Generation of a triple‐gene knockout mammalian cell line using engineered zinc‐finger nucleases . Biotechnol Bioeng 2010 ; 106 : 97 – 105 . Google Scholar PubMed OpenURL Placeholder Text WorldCat 54 Ruis BL , Fattah KR, Hendrickson EA. The catalytic subunit of DNA‐dependent protein kinase regulates proliferation, telomere length, and genomic stability in human somatic cells . Mol Cell Biol 2008 ; 28 : 6182 – 6195 . Google Scholar Crossref Search ADS PubMed WorldCat 55 Brown JP , Wei W, Sedivy JM. Bypass of senescence after disruption of p21CIP1/WAF1 gene in normal diploid human fibroblasts . Science 1997 ; 277 : 831 – 834 . Google Scholar Crossref Search ADS PubMed WorldCat 56 Hannon GJ . RNA interference . Nature 2002 ; 418 : 244 – 251 . Google Scholar Crossref Search ADS PubMed WorldCat 57 Moynahan ME , Jasin M. Mitotic homologous recombination maintains genomic stability and suppresses tumorigenesis . Nat Rev Mol Cell Biol 2010 ; 11 : 196 – 207 . Google Scholar Crossref Search ADS PubMed WorldCat 58 Moehle EA , Rock JM, Lee YL et al. Targeted gene addition into a specified location in the human genome using designed zinc finger nucleases . Proc Natl Acad Sci USA 2007 ; 104 : 3055 – 3060 . Google Scholar Crossref Search ADS PubMed WorldCat 59 Porteus MH , Baltimore D. Chimeric nucleases stimulate gene targeting in human cells . Science 2003 ; 300 : 763 . Google Scholar Crossref Search ADS PubMed WorldCat 60 Goldberg AD , Banaszynski LA, Noh KM et al. Distinct factors control histone variant H3.3 localization at specific genomic regions . Cell 2010 ; 140 : 678 – 691 . Google Scholar Crossref Search ADS PubMed WorldCat 61 Porteus MH . Mammalian gene targeting with designed zinc finger nucleases . Mol Ther 2006 ; 13 : 438 – 446 . Google Scholar Crossref Search ADS PubMed WorldCat 62 Naldini L , Blomer U, Gallay P et al. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector . Science 1996 ; 272 : 263 – 267 . Google Scholar Crossref Search ADS PubMed WorldCat 63 Nightingale SJ , Hollis RP, Pepper KA et al. Transient gene expression by nonintegrating lentiviral vectors . Mol Ther 2006 ; 13 : 1121 – 1132 . Google Scholar Crossref Search ADS PubMed WorldCat 64 Philippe S , Sarkis C, Barkats M et al. Lentiviral vectors with a defective integrase allow efficient and sustained transgene expression in vitro and in vivo . Proc Natl Acad Sci USA 2006 ; 103 : 17684 – 17689 . Google Scholar Crossref Search ADS PubMed WorldCat 65 Vargas J , Jr, Gusella GL, Najfeld V et al. Novel integrase‐defective lentiviral episomal vectors for gene transfer . Hum Gene Ther 2004 ; 15 : 361 – 372 . Google Scholar Crossref Search ADS PubMed WorldCat 66 Yanez‐Munoz RJ , Balaggan KS, MacNeil A et al. Effective gene therapy with nonintegrating lentiviral vectors . Nat Med 2006 ; 12 : 348 – 353 . Google Scholar Crossref Search ADS PubMed WorldCat 67 Lim JK , Glass WG, McDermott DH et al. CCR5: No longer a “good for nothing” gene–chemokine control of West Nile virus infection . Trends Immunol 2006 ; 27 : 308 – 312 . Google Scholar Crossref Search ADS PubMed WorldCat 68 Smith JR , Maguire S, Davis LA et al. Robust, persistent transgene expression in human embryonic stem cells is achieved with AAVS1‐targeted integration . Stem Cells 2008 ; 26 : 496 – 504 . Google Scholar Crossref Search ADS PubMed WorldCat 69 Kotin RM , Linden RM, Berns KI. Characterization of a preferred site on human chromosome 19q for integration of adeno‐associated virus DNA by non‐homologous recombination . EMBO J 1992 ; 11 : 5071 – 5078 . Google Scholar Crossref Search ADS PubMed WorldCat 70 Tan I , Ng CH, Lim L et al. Phosphorylation of a novel myosin binding subunit of protein phosphatase 1 reveals a conserved mechanism in the regulation of actin cytoskeleton . J Biol Chem 2001 ; 276 : 21209 – 21216 . Google Scholar Crossref Search ADS PubMed WorldCat 71 Sauer B , Henderson N. Site‐specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage P1 . Proc Natl Acad Sci USA 1988 ; 85 : 5166 – 5170 . Google Scholar Crossref Search ADS PubMed WorldCat 72 Kohli M , Rago C, Lengauer C et al. Facile methods for generating human somatic cell gene knockouts using recombinant adeno‐associated viruses . Nucleic Acids Res 2004 ; 32 : e3 . Google Scholar Crossref Search ADS PubMed WorldCat 73 Elbashir SM , Harborth J, Lendeckel W et al. Duplexes of 21‐nucleotide RNAs mediate RNA interference in cultured mammalian cells . Nature 2001 ; 411 : 494 – 498 . Google Scholar Crossref Search ADS PubMed WorldCat 74 Hannon GJ , Rossi JJ. Unlocking the potential of the human genome with RNA interference . Nature 2004 ; 431 : 371 – 378 . Google Scholar Crossref Search ADS PubMed WorldCat 75 Ren YX , Finckenstein FG, Abdueva DA et al. Mouse mesenchymal stem cells expressing PAX‐FKHR form alveolar rhabdomyosarcomas by cooperating with secondary mutations . Cancer Res 2008 ; 68 : 6587 – 6597 . Google Scholar Crossref Search ADS PubMed WorldCat 76 Riggi N , Suva ML, Suva D et al. EWS‐FLI‐1 expression triggers a Ewing's sarcoma initiation program in primary human mesenchymal stem cells . Cancer Res 2008 ; 68 : 2176 – 2185 . Google Scholar Crossref Search ADS PubMed WorldCat 77 Montserrat N , Garreta Bahima E, Gonzalez F et al. Simple generation of human induced Pluripotent stem cells using Poly({beta}‐Amino Esters) as non‐viral gene delivery system . J Biol Chem 2011 ; 286 : 12417 – 12428 . Google Scholar Crossref Search ADS PubMed WorldCat 78 Elliott B , Jasin M. Double‐strand breaks and translocations in cancer . Cell Mol Life Sci 2002 ; 59 : 373 – 385 . Google Scholar Crossref Search ADS PubMed WorldCat 79 Richardson C , Jasin M. Frequent chromosomal translocations induced by DNA double‐strand breaks . Nature 2000 ; 405 : 697 – 700 . Google Scholar Crossref Search ADS PubMed WorldCat 80 Szczepek M , Brondani V, Buchel J et al. Structure‐based redesign of the dimerization interface reduces the toxicity of zinc‐finger nucleases . Nat Biotechnol 2007 ; 25 : 786 – 793 . Google Scholar Crossref Search ADS PubMed WorldCat 81 Maeder ML , Thibodeau‐Beganny S, Osiak A et al. Rapid “open‐source” engineering of customized zinc‐finger nucleases for highly efficient gene modification . Mol Cell 2008 ; 31 : 294 – 301 . Google Scholar Crossref Search ADS PubMed WorldCat 82 Blackwell TK , Weintraub H. Differences and similarities in DNA‐binding preferences of MyoD and E2A protein complexes revealed by binding site selection . Science 1990 ; 250 : 1104 – 1110 . Google Scholar Crossref Search ADS PubMed WorldCat 83 Tuerk C , Gold L. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase . Science 1990 ; 249 : 505 – 510 . Google Scholar Crossref Search ADS PubMed WorldCat 84 Phillips CM , Meng X, Zhang L et al. Identification of chromosome sequence motifs that mediate meiotic pairing and synapsis in C. elegans . Nat Cell Biol 2009 ; 11 : 934 – 942 . Google Scholar Crossref Search ADS PubMed WorldCat 85 Shukla VK , Doyon Y, Miller JC et al. Precise genome modification in the crop species Zea mays using zinc‐finger nucleases . Nature 2009 ; 459 : 437 – 441 . Google Scholar Crossref Search ADS PubMed WorldCat 86 Bozas A , Beumer KJ, Trautman JK et al. Genetic analysis of zinc‐finger nuclease‐induced gene targeting in Drosophila . Genetics 2009 ; 182 : 641 – 651 . Google Scholar Crossref Search ADS PubMed WorldCat 87 Lowry WE , Richter L, Yachechko R et al. Generation of human induced pluripotent stem cells from dermal fibroblasts . Proc Natl Acad Sci USA 2008 ; 105 : 2883 – 2888 . Google Scholar Crossref Search ADS PubMed WorldCat 88 Mali P , Ye Z, Hommond HH et al. Improved efficiency and pace of generating induced pluripotent stem cells from human adult and fetal fibroblasts . Stem Cells 2008 ; 26 : 1998 – 2005 . Google Scholar Crossref Search ADS PubMed WorldCat 89 Park IH , Zhao R, West JA et al. Reprogramming of human somatic cells to pluripotency with defined factors . Nature 2008 ; 451 : 141 – 146 . Google Scholar Crossref Search ADS PubMed WorldCat 90 Takahashi K , Tanabe K, Ohnuki M et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors . Cell 2007 ; 131 : 861 – 872 . Google Scholar Crossref Search ADS PubMed WorldCat 91 Yu J , Vodyanik MA, Smuga‐Otto K et al. Induced pluripotent stem cell lines derived from human somatic cells . Science 2007 ; 318 : 1917 – 1920 . Google Scholar Crossref Search ADS PubMed WorldCat 92 Hendrie PC , Russell DW. Gene targeting with viral vectors . Mol Ther 2005 ; 12 : 9 – 17 . Google Scholar Crossref Search ADS PubMed WorldCat 93 Baum C , von Kalle C, Staal FJ et al. Chance or necessity? Insertional mutagenesis in gene therapy and its consequences . Mol Ther 2004 ; 9 : 5 – 13 . Google Scholar Crossref Search ADS PubMed WorldCat 94 Bushman F , Lewinski M, Ciuffi A et al. Genome‐wide analysis of retroviral DNA integration . Nat Rev Microbiol 2005 ; 3 : 848 – 858 . Google Scholar Crossref Search ADS PubMed WorldCat 95 Montini E , Cesana D, Schmidt M et al. Hematopoietic stem cell gene transfer in a tumor‐prone mouse model uncovers low genotoxicity of lentiviral vector integration . Nat Biotechnol 2006 ; 24 : 687 – 696 . Google Scholar Crossref Search ADS PubMed WorldCat 96 Nienhuis AW , Dunbar CE, Sorrentino BP. Genotoxicity of retroviral integration in hematopoietic cells . Mol Ther 2006 ; 13 : 1031 – 1049 . Google Scholar Crossref Search ADS PubMed WorldCat 97 Hacein‐Bey‐Abina S , Von Kalle C, Schmidt M et al. LMO2‐associated clonal T cell proliferation in two patients after gene therapy for SCID‐X1 . Science 2003 ; 302 : 415 – 419 . Google Scholar Crossref Search ADS PubMed WorldCat 98 Hanna J , Wernig M, Markoulaki S et al. Treatment of sickle cell anemia mouse model with iPS cells generated from autologous skin . Science 2007 ; 318 : 1920 – 1923 . Google Scholar Crossref Search ADS PubMed WorldCat 99 Raya A , Rodriguez‐Piza I, Guenechea G et al. Disease‐corrected haematopoietic progenitors from Fanconi anaemia induced pluripotent stem cells . Nature 2009 ; 460 : 53 – 59 . Google Scholar Crossref Search ADS PubMed WorldCat Author notes Author contributions: J.C.: contributed to the conception and writing of the manuscript and final approval; M.L.: contributed to conception and writing of the manuscript, final approval and fund raising. Disclosure of potential conflicts of interest is found at the end of this article. First published online in STEM CELLS EXPRESS May 4, 2011. Telephone: 44‐191‐241‐8688; Fax: 44‐191‐241‐8666 Copyright © 2011 AlphaMed Press This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - Concise Review: Putting a Finger on Stem Cell Biology: Zinc Finger Nuclease‐Driven Targeted Genetic Editing in Human Pluripotent Stem Cells
JF - Stem Cells
DO - 10.1002/stem.658
DA - 2011-07-01
UR - https://www.deepdyve.com/lp/oxford-university-press/concise-review-putting-a-finger-on-stem-cell-biology-zinc-finger-w8qlSNdJbA
SP - 1021
EP - 1033
VL - 29
IS - 7
DP - DeepDyve
ER -