br Gene editing using ssODNs

Gene editing using ssODNs as donors Although a remaining loxP or FRT site may in many cases not influence endogenous gene regulation, and the excision of a PB transposon in most cases leads to restoration of the WT sequence, footprintless gene correction using ssODNs as donors is considered as the more universal and elegant approach. The possibility to correct disease-specific mutations or to introduce specific mutations in patient-specific iPSCs provides entirely new opportunities in disease modelling, drug screening and last but not least individualized cell therapies. IPSC lines engineered in such ways represent perfect isogenic controls for in vitro assays and screens, and may enable safe cellular therapies for many genetic diseases. In addition to correction or insertion of mutations, homologous recombination with ssODNs as donors also allows for the insertion of short modifications such as protein tags or recombination sites into endogenous loci where additional selective elements would be unfavourable. Hence, for many purposes, the integration of small modifications using designer nucleases and ssODNs without any selection markers is the tool of choice. Therefore, Fig. 5 exemplarily illustrates the workflow for a SNP conversion using ssODNs, recombinant Cas9 protein and in vitro transcribed guide RNA. Compared to antibiotic selection-based targeting, a ssODN approach obviates time-consuming donor plasmid generation and a second round of single cell cloning after transgene excision. Furthermore, there is also the possibility for enrichment of nuclease expressing cells via FACS if the transfected vectors contain a fluorescent reporter. In general, it can be expected that the targeting efficiency is higher when using ssODNs due to a higher amount of donor template in the cell which increases the likelihood for pairing with genomic sequences. Furthermore, it is expected that the sequence modulation by a ssODN is operative throughout the whole cell cycle, in contrast to double stranded donors which enter the classic HR pathways that are only active during the S and G2 phase of the order Microcystin-LR (Liu et al., 2010; Radecke et al., 2006; Schubert et al., 2011). Meanwhile, several groups, including ourselves, have shown selection-independent clone generation by using designer nucleases and ssODNs in human PSCs (see Supplementary Table 1). In some studies an initial cell sorting-based enrichment step of nuclease expressing cells prior to subsequent PCR-based screening of the enriched cell population was included (Ding et al., 2013a; Ding et al., 2013b; Soldner et al., 2011) whereas others identified correctly targeted clones without any pre-selection (Merkert et al., 2014; Yang et al., 2013). Miyaoka and colleagues applied the recently developed droplet digital PCR (ddPCR) to capture even rare mutational events in order to generate footprintless iPSC lines with precise mutations (Miyaoka et al., 2014). Further improved targeting efficiencies would considerably facilitate selection-independent targeting approaches in PSCs. Therefore, small molecules that effectively activate or block certain DNA repair pathways can be used (Chu et al., 2015; Maruyama et al., 2015; Yu et al., 2015). For the design of the ssODNs we recommend a maximum length of 120bp with two homology arms of 50bp each, as illustrated in Fig. 5. The total homology length should be at least 40bp (Chen et al., 2011a) and has to exactly match the target genomic sequence because of its shortness. Thus, confirmation of the respective genomic sequence in the targeted cell line is essential prior to the design of the ssODN. In addition to the base pairs of interest which are included in the ssODN, we also recommend the integration of additional silent mutations to increase the accuracy of a targeting specific primer for screening purposes. In general, the diagnostic targeting PCR should run as robust as possible since it is the essential step for identifying targeted cell clones. Thus, it is worth it to test different polymerases, primer pairs and PCR conditions to assess the optimal settings. Of course, it is helpful to have a positive control template for PCR establishment, but often this is only available after successful targeting. Therefore we recommend the analysis of the targeted cell population on day 2 or day 3 after transfection since this should definitely comprise targeting events, if targeting was successful. Regardless, this primary analysis should always be performed to decide whether it is worth it to continue with the elaborate limiting dilution and screening procedure. During screening, the positive control is again essential to exclude discarding false negative pools or clones due to PCR problems. For the screening of hundreds of clones we also recommend the application of cell lysis buffer which can be applied directly into the PCR without elaborate genomic DNA isolation and any washing and precipitation steps. Direct PCR lysis reagents as well as the application of polymerase reaction buffers including loading dyes substantially simplify the entire screening process and considerably facilitate the PCR-based screening process.