br Discussion However it is important
Discussion However, it is important to note that a number of studies have demonstrated that iPS cell lines derived from skin biopsies typically harbor a unique subset of de novo genetic abnormalities, either in the form of copy-number variation or single base-pair changes (Abyzov et al., 2012; Gore et al., 2011) and that iPS cell lines generated from the same parental line can vary significantly with respect to whole-genome gene expression in the differentiated state (Reinhardt et al., 2013). Nonetheless, it is reasonable to expect that the confounding effects arising from the variations that exist across different iPS cell clones may be minimized by comparing multiple gene-corrected or gene-targeted clones with multiple uncorrected clones. In this regard a consistent difference that is observed exclusively in the corrected versus uncorrected lines can most likely be attributed to the patient-specific mutation rather than variations that may exist from one clone to the next. In the current study we routinely observed targeting efficiencies of > 5%, enabling the generation of multiple gene-targeted and “matched” uncorrected clones from a single experiment. The relatively high gene-targeting frequencies obtained using our one-step protocol may in part be attributed to the possibility that the iPS cell colonies themselves act as a form of selection. This is based on the assumption than an iPS cell colony that has taken up the plasmids required for reprogramming will have also taken up the DNA constructs required for gene targeting. Following simultaneous reprogramming and targeting of the DNMT3B locus in the fibroblast line derived from a patient with retinitis pigmentosa, the estimated targeting efficiency in the total iPS cell population was approximately 5%, more than 5-fold higher than that observed in the embryonic stem cell line H9. With respect to the ADA and PRPF8 gene correction experiments, we obtained targeting efficiencies of 5% and 8%, respectively. This is notably higher than the frequencies of gene targeting that have previously been reported in pluripotent stem DMXAA using ssODNs, without the aid of drug selection, where correctly targeted cells typically comprise less than 1% of the total population (Soldner et al., 2011; Yang et al., 2013). Another notable advantage of our one-step protocol over conventional approaches is that it does not require additional steps for the clonal isolation of iPS cells. In the absence of selection, this is most often performed using fluorescently activated cell sorting or limiting cell dilution to expand a clonal population from a single iPS cell, which is inefficient and cumbersome because human pluripotent stem cells exhibit poor survivability in the absence of appropriate cell-to-cell contacts. Although bi-allelic Cas9-induced modification was a common outcome in the iPS cell lines generated in this study, we show that this can be minimized by designing sgRNAs that specifically target patient-specific mutations. This approach is feasible for patients with autosomal dominant and compound heterozygous mutations, but not for mutations that are autosomal recessive. However, since autosomal recessive diseases normally require only a single functional allele, bi-allelic modification resulting in correction of one allele and mutagenic NHEJ in the other, is less of a concern. It will also be important to evaluate the use of Cas9 variants that have improved DNA cleavage specificity in the protocol described here. Use of the paired nickase (Cas9-D10A) (Mali et al., 2013a; Ran et al., 2013) and dCas9-Fok1 fusions (fCas9) (Guilinger et al., 2014), for example, should minimize the potential of off-target effects that are largely associated with the wild-type form of the Cas9 protein. Keeping background damage to a minimum will be essential not only for retaining the downstream therapeutic potential of the cells but also for an accurate recapitulation of the disease and corrected (wild-type) phenotype, which will be important for disease modeling and drug discovery purposes. Finally, there is also significant value in adapting our protocol to permit the generation of gene-corrected iPS cell lines from alternative cell sources such as blood, which involve less invasive procedures and may harbor fewer somatic mutations compared with fibroblasts derived from skin biopsies. Since expansion of fibroblasts from an initial skin biopsy can take several weeks, the ability to simultaneously reprogram and genetically repair cells isolated from blood draws will also serve to expedite the process of generating gene-corrected iPS cell lines even further.