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    • Although in previous studies by Soldner et

    2018-11-08

    Although in previous studies by Soldner et al. (2011) and Ding et al. (2013), an initial FACS-based enrichment step of TALEN-expressing ibuprofen msds prior to subsequent PCR-based screening of this enriched cell population had to be included, both groups provided initial evidence that the footprintless correction of disease-specific mutations in hPSCs without the need for an antibiotic selection is generally possible through the application of single-stranded oligodeoxynucleotides (ssODNs). In this study, we report on an efficient technique for nonviral and selection-independent gene targeting in hESCs and hiPSCs. For the development of our targeting protocol, we first used an iPSC enhanced GFP (eGFP) reporter system in which ZFNs were applied to disrupt the eGFP open reading frame by NHEJ with an overall efficiency of up to 4%, and to integrate a red fluorescent protein into the eGFP locus by HR. Without any antibiotic selection, we achieved HR-targeting efficiencies of up to 1.2% and could show that the ZFN-treated PSCs preserved their pluripotency and chromosomal integrity. Finally, we targeted the endogenous “safe harbor” locus AAVS1, which is known for robust transgene expression in PSCs (Smith et al., 2008). Here, by using ZFNs and TALENs, we obtained targeting efficiencies comparable to those achieved with our eGFP/RedStar reporter system for one hESC and two hiPSC lines, and generated stable transgenic PSC lines by FACS. Moreover, by applying TALENs together with short ssODN donors without any preselection, we show that the high targeting efficiencies obtained actually facilitate direct PCR screening of correctly targeted clones. This should enable footprintless gene correction and transgene-independent isolation of mutation-corrected, disease-specific iPSC clones, and may ultimately lead to novel concepts for iPSC-based ex vivo gene therapies.
    Results
    Discussion Genetic engineering techniques based on tailored nucleases, such as ZFNs and TALENs, represent a genuine breakthrough in gene targeting. In particular, these technologies offer exciting opportunities for genetic engineering in patient-specific iPSCs with respect to the introduction of reporter genes as well as the correction and introduction of disease-specific mutations. Apart from two studies (Ding et al., 2013; Soldner et al., 2011), all known publications in this area have applied transgene-based antibiotic selection to isolate targeted cell clones. Obviously, low efficiencies typically continue to hamper the antibiotic-selection-free isolation of correctly targeted clones and impede footprintless correction and introduction of disease-specific mutations. For the establishment and optimization of our targeting protocol, we used a reporter iPSC line, which enabled us to visualize successful ZFN-based gene targeting in hPSCs. Using this line, we observed a proportion of up to 4% of eGFPneg cells 6 days after transfection, which is similar to what was recently reported for NHEJ-based dTomato reporter inactivation in the active DNMT3b locus in hESCs by using CRISPR RNA-guided nucleases (Hou et al., 2013). Remarkably, the observed proportion of eGFPneg cells does not reflect the actual efficiency of DSB generation. Targeted DSBs are probably induced in a considerably higher proportion of cells, followed by an unknown frequency of error-free repair by NHEJ. In order to extend these findings to HR-based gene targeting, we constructed a donor plasmid with two arms of ∼700 bp of DNA homologous to the sequences enclosing the predicted ZFN target site within the genomically inserted eGFP. Notably, a red fluorescence reporter, RedStarnuc, which is targeted to the nuclear membrane, was chosen in order to ensure discrimination from potential autofluorescent cells. Also of importance, the RedStarnuc was not coupled to a promoter, but was cloned in frame to the eGFP sequence via a 2A sequence to exclude transient, plasmid-based RedStar expression, and to minimize a potential background due to undesired reporter expression after random genomic insertion of the donor plasmid. Three days after transfection of the hCBiPS2eGFPC7 reporter line with the ZFN plasmids and the donor plasmid, a population of up to 1.2% of RedStarnucpos cells was detectable. At this time point, the vast majority of these cells still expressed eGFP, which can be explained by the typical persistence of eGFP mRNA/protein in hPSCs for several days (Hartung et al., 2013). Also, this persistence over a period of at least 3 days correlates perfectly with our observation of the time course of fluorescence loss after NHEJ-based eGFP inactivation. Six days after transfection, however, no eGFPposRedStarpos cells were observed, indicating that almost all RedStarpos cells represent correctly targeted cells. The fact that cell clusters consisting of eGFPneg RedStarpos cells were distributed over the whole plate speaks strongly in favor of a multitude of independent HR events. In this way, we achieved considerably higher targeting rates than those previously reported for the correction of a mutant eGFP reporter gene in 0.24% of hESCs and 0.14% of hiPSCs (Zou et al., 2009).