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  • One important application of designer nucleases in

    2018-11-06

    One important application of designer nucleases in human PSCs is the targeted integration of transgenes at specific genomic loci that enable robust expression not only in undifferentiated cells but also in the differentiated derivatives. So-called ‘safe harbour sites’ support more or less stable levels of transgene expression from the external promoters of choice and can be located intragenic in introns or extragenic. They are considered as safe because integration of transgenes apparently does not lead to oncogenic transformation or show any signs of genotoxic effects (Sadelain et al., 2012). As yet, the AAVS1 and CCR5 safe harbour loci are most frequently used, whereby the last one has the attractive side effect of providing resistance of hematopoietic cell lineages to HIV-transduction (Perez et al., 2008). According to own experience, especially targeting of AAVS1 is highly efficient in hPSCs with relative targeting frequencies of more than 1% (Merkert et al., 2014). Specific and efficient TALENs as well as guide RNAs have been published for both sites and can be obtained from academic sources or can be designed accordingly. Although according to literature constitutively expressed fluorescence markers represent the most frequent transgenes inserted into safe harbour sites, single cell cloning is in most cases based on additional AMD3100 genes (see Supplemental Table 1). Clearly, the application of selection cassettes simplifies the clone generation and is typically without adverse effects as long as the cell lines are not produced for later clinical use. Regulated transgene expression from a safe harbour site can be achieved via inducible expression systems or tissue-specific promoters enabling for example the isolation of specific cell populations (Gantz et al., 2012; Hockemeyer et al., 2009; Qian et al., 2014; Tiyaboonchai et al., 2014). Constitutive promoters provide reliable stable transgene expression, e.g., allowing functional correction of genetic diseases (Chang and Bouhassira, 2012; Zou et al., 2011b) or reporter gene-based molecular imaging for transplantation experiments (Wang et al., 2012). If a well-characterized promoter fragment is available, even tissue- or cell type-specific transgene expression can be realized from safe harbour loci. Due to the availability of established designer nucleases and donor plasmids with homology arms for common safe harbour loci, integration of potentially cell type-specific reporter cassettes into such loci with subsequent testing of its specificity in differentiated hPSC derivatives is often easily accomplished. If for a given promoter fragment such an approach does not result in sufficient expression levels or adequate cell type-specificity, the much more laborious strategy of integrating reporter or selection genes into an endogenous gene locus of interest has to be considered. In this case, new nucleases and donor plasmids for the respective locus typically have to be generated. The design of both, nuclease and donor plasmid, critically depends on the question whether it is acceptable that one allele of the affected gene is functionally destroyed or whether it is crucial to maintain expression of both gene alleles. There are different possibilities to preserve a functional genomic allele, like transgene integration into intron sequences or the generation of fusion proteins. The latter was applied for example for OCT4 reporter cell lines monitoring the pluripotency state of hPSC cultures (Hockemeyer et al., 2009; Hockemeyer et al., 2011; Hou et al., 2013). In our experience, reporter gene integration replacing the endogenous stop codon in combination with a self-cleaving 2A peptide is the approach of choice to maintain the endogenous gene function. Fig. 4 illustrates the use of such a 2A site which should result in the expression of both, the endogenous and the reporter protein under control of the physiological promoter. Finally, the decision for a specific targeting strategy always depends on the transcriptional regulation of the gene of interest and the formation of different splice variants.