For instance, one study showed that following transfection of 2.46108 cells with a targeting vector containing large homology arms, antibiotic selection and PCR screening led to the isolation of two targeted cell clones. We obtained a,0.1% gene correction rate using engineered nucleases. Thus, the induction of a site specific double-strand break increased the targeting frequency by.100,000 fold. The low rate of gene correction in SSCs may reflect inherent mechanisms of genome protection unique to germ cells; intrinsic differences in efficiency between cell types are not unexpected. Fortunately, one could invoke a strategy to enrich for corrected cells using multiple published methods. Enrichment strategies include FACS purification of transfected cells, the purification of cells that have undergone correction based on the modification of a surrogate reporter which dramatically enriches for modified cells, and the use of donor constructs and designs containing selectable markers that allow one to select for modified cells using the selectable marker and then subsequent “scarless” elimination of the selectable marker after identification. Thus, the low frequency of gene correction in SSCs does not preclude genome editing from being accomplished in this important stem cell type. The implications of our study are multi-faceted with applications in research and potentially ICG-001 therapeutics. Much progress remains to be made in understanding mechanisms controlling SSC fate, particularly in humans. The ability to make precise modifications to the genome could facilitate analysis of gene function, thereby advancing our understanding of SSCs and spermatogenesis. For instance, a point mutation identified in a genome-wide association study to be potentially associated with spermatogenic failure could be directly tested for functional importance using the technology demonstrated here. The ability to generate fluorescent reporters of gene expression by targeted addition is another potential research application. The relevance of these applications extends even beyond the study of SSCs, given that upon testicular transplantation of genetically engineered GS cells new transgenic mouse models can be generated. The implications of our study for medicine are two-fold. First, we addressed a pervasive challenge in gene therapy, namely gene delivery in a “hard to transfect” primary-like stem cell. Gene delivery is a particularly significant issue for nuclease-mediated gene correction because it is necessary to introduce three components into cells. In this study we demonstrated a gene delivery approach that may be widely applicable to other stem cells. In our preliminary experiments adeno-associated virus, and integration deficient lentivirus both were inadequate for accomplishing genome editing. Identification of a virus with the appropriate tropism for a cell type of interest and production of sufficient titers of infectious virus are among the complications of viral delivery. In contrast, following brief optimization experiments, we found the Neon electroporator could impart unprecedented high transfection rates with GS cells. Further, the approach can be applied on both a small and large scale, allowing for cell-type specific optimization experiments.