A modified TALEN-based system for robust generation of knock-out human pluripotent stem cell lines and disease models
© Frank et al.; licensee BioMed Central Ltd. 2013
Received: 24 June 2013
Accepted: 7 November 2013
Published: 9 November 2013
Transcription activator-like effector nucleases (TALENs) have emerged as a tool for enabling targeted gene editing and disruption in difficult systems, such as human pluripotent stem cells (hPSCs). The modular architecture of TAL effectors theoretically enables targeting of any genomic locus and several cloning systems for custom TALEN assembly have recently been established. However, there is a lack of versatile TALEN expression systems applicable to hPSCs.
Here, we extend an existing TALE assembly system by a dual set of expression vectors for efficient application of TALEN technology in hPSCs. This is characterized by improved TALEN architecture as well as antibiotic resistance and fluorescent reporter cassettes, thus enabling enrichment for transfected cells.
Improved functionality of the combined system was demonstrated by targeted disruption of the HPRT1 gene to create isogenic disease models of Lesch-Nyhan-Syndrome. Using female hPSCs, homozygous disruption of HPRT1 occurred at efficiencies of up to 15%. Differentiating isogenic knock-out cells both into central nervous system (CNS) as well as into sensory-like neurons recapitulated previously described phenotypes based on patient-specific induced PSCs and extended these findings to non-CNS neurons, respectively.
The combined vector system allows for flexible and affordable generation of knock-out hPSCs lines, thus enabling investigation of developmental processes as well as the generation of isogenic disease models without the need for patient material.
KeywordsHuman pluripotent stem cells Targeted gene disruption TALE nucleases Disease modeling
Gene targeting in human pluripotent stem cells (hPSCs) by conventional approaches is a cumbersome and inefficient process. The development of sequence-specific nucleases, such as TALENs, can, however, significantly enhance the efficiency of genome editing in hPSCs . TALENs are chimeric fusions between custom-designed transcription activator-like effectors (TALE) of Xanthomonas plant pathogens and the FokI nuclease [2–4]. Within the TALE structure, individual repeat domains confer specific recognition and binding to single nucleotides on DNA. Several types of repeat domains differing solely in their so-called repeat variable di-residues (RVDs) have been found to be selective binders of individual DNA bases, with differing affinities . Custom design of the modular TALE repeat domain structure hence allows specific targeting and binding of TALEs to genomic regions of interest. Upon presence and adjacent binding of two TALENs, a DNA double-strand break (DSB) will be induced by the fused catalytic domain of FokI nuclease, which is then repaired either by the error-prone mechanism of non-homologous end joining (NHEJ) or via homology-directed repair . Hence, in the absence of homologous template sequence, small genetic lesions may be introduced into a predefined locus by delivery of pairs of specifically designed TALEN constructs into cells, such as hPSCs [1, 7].
Several approaches have been used for the generation of custom TALENs [7–11]. These are, however, not easy to adopt for new researchers entering the field . Cermak et al. have recently established a particularly straightforward TALEN assembly system that is based on GoldenGate cloning. It enables reliable TALEN assembly within a few days and has been made available through the Addgene repository. However, the associated expression vectors were not optimized for applications in mammalian cells. We have therefore developed a new set of vectors compatible with this publicly available TALEN assembly kit . Our plasmids contain an improved TALEN backbone architecture incorporating findings by Miller et al. as well as selection cassettes to enrich for transfected cells. Here, we report the application of the combined system to hPSCs by creating isogenic knock-out models for the X-linked HPRT1 locus at high efficiencies. Mutations in this gene cause Lesch-Nyhan-Syndrome (LNS), a disease with strong neurological symptoms [14, 15]. Clonal knock-out lines showed impaired differentiation into different neuronal lineages, recapitulating aspects of the disease phenotype in vitro. The combined TALEN assembly-and-expression system simplifies the custom generation of hPSC knock-out cell lines and will therefore be universally applicable for functional studies and the generation of hPSC-based disease models.
hPSCs were cultivated on Matrigel® in either MEF-conditioned or defined FTDA media [16, 17]. HEK-293 T cells were cultured on conventional tissue culture plastic in DMEM supplemented with 10% fetal bovine serum, non-essential amino acids, 2-mercaptoethanol, and Penicillin-Streptomycin-L-Glutamine.
TALEN design and assembly
TALE repeat structures were designed using either the ZiFit targeter http://zifit.partners.org/ZiFiT/) or the TAL Effector Nucleotide Targeter 2.0 (https://tale-nt.cac.cornell.edu/node/add/talen). TALENs were assembled as published , by following the protocol associated with the GoldenGate TALEN and TAL Effector Kit 1.0 (Addgene #1000000016), except that TALE repeats were ultimately cloned into vectors pTAL7A (for TALEN A) and pTAL7B (for TALEN B) (Additional file 1: Figure S1).
Pretesting of TALENs and generation of HPRT1 knock-out lines
TALENs A and B were transfected into HEK-293 T cells or hPSCs at equimolar ratios using Fugene 6 (Roche). Starting from 24 hours after selection, antibiotic selection was applied for 48 hours with puromycin at 0.5 μg/ml and blasticidin at 5 μg/ml. After selection, cells were dissociated and (i) lysed to purify genomic DNA, (ii) analyzed via flow cytometry, or (iii) reseeded for selection of knock-out cells using 6-thioguanine (6-TG, Sigma, #A4862). Mutation frequencies were determined with the CEL-1 assay (Surveyor Nuclease S, Transgenomic, #706020) according to the manufacturer’s protocol. 6-TG was applied for 4–8 days at a concentration of 30 μM.
Differentiation of hPSCs into neurons was performed as previously described [18, 19]. Quantification of neurite length and percentage of beta-III-tubulin-positive neurons was performed using Arrayscan XTI HCA high-content imaging instrumentation (Thermo).
Availability of supporting data
The data sets supporting the results of this article are available in the European Nucleotide Archive, IDs HG530137 and HG530138, http://www.ebi.ac.uk/ena/, as well as in the Addgene plasmid repository, IDs 48705 and 48706, http://www.addgene.org/.
Comparison of pTAL4 and pTAL7 vector systems
Site for GoldenGate TALEN Assembly
Screening for successful cloning
Full length Voytas et al. 
Truncated C- & N-termini similar to Miller et al. 
Mammalian cells (CAG promoter + chimeric intron + Kozak sequence)
Enrichment of transfected cells
GFP & Puromycin (pTAL7A) Blasticidin (pTAL7B)
Not optimized for mammalian cells
Optimized for mammalian cells
To demonstrate the functionality of the combined system, we designed 3 pairs of TALENs targeting exon 2 of the HPRT1 gene (Additional file 1: Table S1). HPRT1 is located on the X chromosome and mutations in this gene cause Lesch-Nyhan-Syndrome (LNS), a disease with strong neurological symptoms [14, 15]. Cells without functional HPRT1 can be selected via 6-thioguanine, a guanine analogue that is metabolized by HPRT1 and introduced into the DNA, resulting in mutagenesis and cell death (Figure 1B). Robust expression of cloned TALEN and selection cassettes in mammalian cells was confirmed by qRT-PCR (Additional file 1: Figure S2A,B). TALEN constructs were once more transfected into 293 T cells, transiently incubated at 37°C and 30°C to also investigate effects of low-temperature incubation on non-homologous end joining (NHEJ)-based mutation frequencies . HPRT1 PCRs on isolated genomic DNA were denatured, reannealed, and subjected to Cel-1 digestion and gel electrophoresis to reveal the generation of small genetic lesions in these bulk cultures. Specificity of DNA-binding of TALENs is mediated by two amino acids in each of the individual repeat domains, the so-called repeat variable di-residue (RVD). Several RVDs have been found to bind with different affinities to their target nucleotide . Using TALENs employing only the “NK” RVD for targeting guanine did not produce detectable Cel-1 digestion fragments (Figure 1B, top panel). However, replacing “NK” by “NN” RVDs at strategic positions, as previously suggested [4, 5], revealed that all three tested TALENs were functional as their delivery into 293 T cells apparently caused robust introduction of small lesions in HPRT1 (Figure 1C, bottom panel, Additional file 1: Table S1). A transient cold-shock at 30°C did not have a significant effect on induction of double-strand breaks (DSBs) (Figure 1C). Functional selection of HPRT1 mutant cells using the 6-TG confirmed these results in that all three TALENs produced 6-TG resistant cells, at varying efficiencies (Figure 1D). Sequencing of PCR clones from these 6-TG selected cultures showed that TALEN delivery mostly resulted in small deletions, implying that resulting frame shifts were the causes of disrupting HPRT1 function (Figure 1E).
We then asked if it was possible to pre-test TALEN pairs directly in hESCs. Indeed, the results were similar in that TALEN pair #2 appeared to cut its target site most efficiently, albeit the obtained Cel-1 signals were somewhat weaker than in 293 T cells (Figure 1F). 6-TG selection functionally confirmed the disruption of the HPRT1 gene in hESCs as numerous resistant colonies appeared with two out of three TALEN pairs tested (Figure 1G). Analysis of the mutational spectrum in hESCs by sequencing of PCR clones revealed that mostly deletions as well as few insertions had been introduced by TALEN delivery, like in 293 T cells (Figure 1H).
Next, we sought to quantify mutation frequencies in hESCs by determining the ratio of surviving 6-TG resistant hESC colony numbers by the total number of colonies emerging in the absence of 6-TG. Using pre-selection of double-transfected hESCs by puromycin and blasticidin, functional HPRT1 mutations were introduced in approximately 15% of cells. Mutation rates without pre-selection were low (0.5%), reflecting the usefulness of these resistance cassettes in the pTAL7 vectors (Figure 2C).
In order to test the applicability of our system to loci other than HPRT1, we employed TALENs targeting the OCT4 as well as the FOXC1 locus. Transfection into hESCs and subsequent analysis showed robust induction of error-prone NHEJ in both these genes (Figure 2D). Moreover, to demonstrate that clonal knock-out hESC lines could be isolated without the option of functional negative selection (as with 6-TG), randomly picked colonies emerging after HPRT1 TALEN transfection were expanded and screened for genomic lesions. Mutant clone screening was in this case performed based on potential disruption of a restriction enzyme recognition site within the TALEN targeting region. Of the 19 clones analyzed, 5 showed an undigested PCR band (Figure 2E) suggesting at least heterozygous mutations. We further analyzed these clones using 6-TG as well as sequencing of the HPRT1 locus. One clone showed small deletions in both alleles and was subsequently functionally confirmed to be 6-TG resistant (#14 in Figure 2E). These data demonstrate the universal applicability of the combined GoldenGate/pTAL7 TALEN system for generating knock-out hESC lines without the need for additional gene targeting vectors or negative selection procedures.
TALENs have become a valuable tool for genome editing in a variety of cell types, including hPSCs [1, 7, 21]. Over the past few years, various methods to assemble TALENs have been developed, ranging from gene synthesis to manual cloning kits and automated high-throughput systems [7–11, 13]. The GoldenGate TALEN assembly kit by Cermak et al. is publicly available, straightforward to establish in the laboratory, reliable, as well as time and cost-efficient . However, the final expression vectors of this kit were not optimized for application in mammalian cells. Furthermore, modifications of the TALEN domain architecture shown to improve DSB induction  were not included in the expression vectors. The pTAL7 vectors described here are fully compatible with the GoldenGate TALEN kit , yet overcome these drawbacks. They enabled robust induction of DSBs in human cell lines, including hPSCs, resulting in functional gene knock-out without the need of conventional targeting vectors. The selection cassettes implemented in the plasmids enable enrichment of double-transfected cells, which proved to be key for obtaining high mutation frequencies in our hands. Pre-selection for double transfectants will particularly be necessary in cases where negative functional selection as in case with HPRT1/6-TG is not an option. Indeed, we demonstrate that the pTAL7 vectors permitted the isolation of hPSC knock-out lines without applying 6-TG selection, based solely on random picking of clones. In comparison with enrichment methods relying on fluorescent marker proteins , the pTAL7 system offers both antibiotic and fluorescent selection of transfected cells, making it highly versatile and independent of the availability of cell sorting instrumentation. Furthermore, conventional lipofection as a delivery method yielded sufficient numbers of clones with small amounts of starting cells, in contrast to methods based on electroporation [1, 7].
The disease phenotypes observed in HPRT1 knock-out cell-derived CNS neurons recapitulated aspects of impaired neurogenesis in LNS patients and were in line with observations made with patient-specific hiPSCs, albeit showing an overall higher number of differentiated neurons in our hands . These differences may be explained with differences among individual hPSC lines, supporting the necessity of isogenic disease models. Future experiments could address the effects of HPRT1 knock-out on neuronal differentiation side-by-side in engineered hESCs and patent-specific hiPSCs. In addition, impaired neurite outgrowth was also observed in BRN3A-positive neurons , extending these findings to the PNS system. Notably, these phenotypes were observed in independent clones of independent cell lines (hESCs & hiPSCs) and in comparison to isogenic parental controls, which demonstrates a causative role of mutant HPRT1 irrespective of the genetic background. It would be worthwhile further investigating this effect on sensory-like neurons to study functional links to LNS phenotypes. Taken together, the observed cellular phenotypes confirm that TALEN-mediated mutagenesis in wild-type hPSCs is a valid alternative for disease modeling without the need for patient material and lengthy reprogramming as well as hiPSC characterization procedures . Introduction of more subtle lesions or gene correction approaches would be enabled by employing conventional gene targeting vectors in addition to the TALEN constructs . Furthermore, the system is not limited to using hPSCs, as we have also successfully used it in HEK-293 T, HeLa, and mouse ES cells (Figure 1C-E, and data not shown).
The field of targeted genetic engineering is rapidly evolving. For example, RNA-protein-mediated DSB induction by the CRISPR-Cas9 system has recently been shown to efficiently enhance gene targeting in a variety of organisms and cell types, including hPSCs, similar to TALENs [22, 24–30]. However, comprehensive studies addressing target specificity of this new platform are yet to be carried out, with regards to the shorter binding sequence of CRISPRs compared to TALENs as well as regarding the tolerance of single-base mismatches in the recognition sequence. Likely, future investigation will be based on several well-working genetic engineering systems that may be selectively employed depending on their respective strengths and weaknesses.
The improved TALEN system evaluated in this work presents an affordable and easy-to-adopt methodology to facilitate gene targeting in human pluripotent stem cells and other mammalian cell types. It will thus be helpful for developmental studies as well as disease modeling approaches.
We thank Dr. Daniel Voytas for making his TALEN assembly system publicly available through Addgene and Dr. Ralf Kühn for providing the pCAG-TAL-linker-IX_Fokwt plasmid. We also thank Peter Reinhardt for advice on the differentiation of CNS neurons and Dr. Susanne Höing for help with high-content imaging analysis. SF also acknowledges the International Max Planck Research School for Molecular Biomedicine (CIM-IMPRS). This work was supported by the Chemical Genomics Centre of the Max Planck Society.
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