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    • In the following we provide an

    2018-11-06

    In the following we provide an overview on recent applications of designer nucleases in human PSCs and on the PI-103 of possible targeting and selection strategies (Supplemental Table 1). Thereby, Fig. 1 highlights the key parameters which have to be considered before starting gene editing and which will be discussed here. Despite the aforementioned limitations concerning a quantitative comparison of the published approaches, we will outline the most suitable approaches for specific applications. In particular, the purpose of this review is to present a practical guide, in terms of experimental considerations, limitations and other critical aspects, for successful gene editing in human PSCs using designer nucleases for various applications.
    Choice and design of the nucleases Fig. 2 briefly illustrates the structure and characteristics of three types of designer nucleases. In summary, ZFNs and TALENs consist of target-specific DNA-binding domains fused to an unspecific nuclease domain, whereby in the CRISPR/Cas9 system a chimeric RNA containing the target sequence guides the Cas9 nuclease to cleave the DNA (Gaj et al., 2013). The development and improvement of customized engineered endonucleases is continuously progressing but efficiencies and grade of specificity of the different nuclease systems are still controversial. TALENs and the CRISPR/Cas9 system have already replaced ZFNs as it is still technically challenging and time-consuming to engineer active ZFNs and only a few academic labs have established routine production (Maeder et al., 2008). The generation of TALENs is much less labour-intensive and time-consuming once the system has been established in the lab, although a typical TALEN requires ~1800bp to be assembled for each new target site. The CRISPR/Cas9 system is the easiest to use, time-saving and relatively cheap, as the synthesis of an only 20bp guide RNA is required to program the nuclease. Undoubtedly, the production of several CRISPR guide RNAs for one genomic locus and the validation of the most effective one is less time-consuming than doing the same for TALENs. On the other hand, it has to be emphasized that in both cases design, construction and preparation of the respective plasmid vectors is usually the minor part of the entire targeting approach compared to the establishment of correctly targeted single cell clones. In general, once the respective systems are established in a lab, the construction can be accomplished in about two weeks for TALENs and in one week for CRISPR/Cas9. Technical guidelines and different TALEN assembly kits (https://www.addgene.org/talen/) as well as different CRISPR cloning systems (https://www.addgene.org/crispr/) are available via Addgene or commercial sources. Online tools that facilitate optimal design of such nucleases and their evaluation concerning efficiency and potential off-target activity can be found on https://tale-nt.cac.cornell.edu/, https://bao.rice.edu/research/, http://www.rgenome.net/cas-offinder/, http://www.genome-engineering.org/ or https://chopchop.rc.fas.harvard.edu/index.php. However, previously validated nucleases are already available from academic and commercial sources for many genomic targets and the effort of designing new ones has to be weighed. After defining the genomic target region, the respective individual DNA sequence should be confirmed to exclude single-nucleotide polymorphisms (SNPs) or other variations in the recognition site of the nuclease. Due to context-dependent interactions between neighbouring zinc-fingers, not all genomic sites can be targeted by ZFNs. In general, one ZFN site can be found every 125–500bp of a random genomic sequence, depending on the assembly method (Kim et al., 2009; Sander et al., 2011; Sander et al., 2010). In contrast, TALENs can be designed for almost any sequence stretch. Merely the presence of a thymine at each 5′ end of the DNA recognition site is required (Mak et al., 2012). In case of the CRISPR guided Cas9 nuclease the presence of a protospacer adjacent motif (PAM) sequence is necessary, which depends on the bacteria species from which the Cas9 was derived (e.g. Streptococcus pyogenes ‘NGG’). The PAM sequence directly downstream of the target sequence is not part of the guide RNA but is obligatory for cutting the DNA strand (Jinek et al., 2012). Due to these sequence requirements, also the design of TALENs and especially of the guide RNA of the CRISPR/Cas9 system may reach limits. This is especially critical when using single stranded oligonucleotides (ssODNs) with short homology arms, which can be applied instead of donor plasmids for footprintless gene correction or the integration of flags or mutations at specific sites of a protein. Thus, under some circumstances the nature of the genomic region as well as the intended application determines the choice of the nuclease.