In our initial attempts at simultaneous

In our initial attempts at simultaneous reprogramming and gene targeting of somatic cells, we chose to target the DNMT3B gene with an EGFP reporter, since DNMT3B is highly expressed in pluripotent Erlotinib Hydrochloride and quickly downregulated following differentiation, allowing targeted iPS cell colonies to be easily identified by fluorescent microscopy. To facilitate homologous recombination at the DNMT3B locus, we used in vitro transcribed mRNA encoding the Cas9 protein derived from N. meningitides (Hou et al., 2013), a plasmid encoding a DNMT3B-specific short-guide (sg)RNA and a donor template encoding an EGFP reporter and puromycin resistance gene flanked by 1-kb homology arms specific to sequences upstream and downstream of the DNMT3B start codon (Figure 2A). We first evaluated targeting of DNMT3B using this system in the embryonic stem cell line H9 and routinely obtained a gene-targeting efficiency of 0.5%–0.9% (Figure 2B). We next co-transfected the reprogramming plasmids along with the DNMT3B-specific gene-targeting factors into a fibroblast line derived from a patient with autosomal dominant retinitis pigmentosa. Although iPS cell colonies first emerged as early as 10 days following transfection, we observed the vast majority emerge between 2 and 3 weeks postelectroporation, at which point the culture was routinely analyzed by fluorescent microscopy. Of three independent experiments we identified 8, 13, and 44 iPS cell colonies that stably expressed the EGFP reporter, indicative of a successful gene-targeting event at the DNMT3B locus (Figure 2C). We obtained a large number of iPS cell colonies (>1,000) from each of these experiments, making it difficult to accurately assess gene-targeting efficiency. Thus, to estimate targeting efficiency in the pool of iPS cells, we passaged cells from a single representative experiment approximately 3 weeks post-transfection using EDTA to selectively remove iPS cells from the residual fibroblasts before re-plating. As measured by the number of EGFP-expressing cells, targeting efficiency was approximately 3% and 5% following flow cytometric analysis of the total cell population after three and five passages, respectively (Figure 2D). An increase in the number of EGFP-expressing cells is most likely due to a further loss of the residual parental fibroblast population, and we did not observe any further increase in the number of EGFP-expressing cells after five passages. Using the reprogramming experiments that were not passaged, we randomly selected and expanded six EGFP-expressing and six EGFP-non-expressing colonies for further analysis. Gene targeting of the DNMT3B locus was confirmed in all six EGFP-expressing clones by PCR using primers that flank the recombination junction site, but not in any of the EGFP-non-expressing clones (Figure 2E). Flow cytometry analysis also revealed a uniform level of EGFP expression in > 95% of the cell population with similar fluorescence intensities observed in all six clones (Figure 2F). Although targeting of DNMT3B in H9 cells with the same donor template routinely yielded numerous puromycin-resistant colonies, confirming functionality of the phosphoglycerate kinase (PGK) promoter in pluripotent stem cells, EGFP-expressing iPS cell lines generated by simultaneous reprogramming and targeting of DNMT3B exhibited puromycin sensitivity. This suggests the PGK promoter was transcriptionally silenced during the reprogramming process, making drug selection of simultaneously reprogrammed and gene-targeted clones infeasible. Conversely, when we used a one-step procedure to generate iPS cell lines with an EGFP reporter fused to the OCT4 coding region using the Cas9/CRISPR system described previously (Hou et al., 2013) (Figure S1), these clones did exhibit resistance to puromycin in the culture media. In this case the puromycin resistance gene is fused to the EGFP reporter via a 2A sequence and is thereby driven from the endogenous OCT4 regulatory region rather than the minimal and non-specific PGK promoter.