We first applied the CTS method to

We first applied the CTS method to a single gene (CRYAB) in hB53 hiPS6 iPSCs (Riedel et al., 2014). Following transfection of a pre-validated CRYAB-specific single guide RNA (sgRNA)-expressing pX330 vector and ssODN donor template (for incorporation of the passenger modification) as well as a commercially available AAVS1-specific TALEN pair and puromycin N-acetyltransferase (pac)-containing donor vector driven by a constitutive promoter (for incorporation of the selectable modification), retinoid x receptor were treated with puromycin per our CTS protocol (to enrich for cells that underwent HDR, Figure S1), for 48 hr (to eliminate untransfected cells), or not at all (to mimic a selection-free system) (Figure S2). Clones were then harvested and analyzed via Sanger sequencing for modification at the gene of interest. The total number of editing events (NHEJ and HDR) was >7-fold greater in CTS cells relative to unselected cells and, crucially, the HDR/NHEJ ratio was >4-fold greater (Figure 1C, left panel, and Table S1). This corresponded to a ∼40% likelihood of picking a precision-modified clone, both heterozygous and homozygous, from the final culture with CTS compared with 2% (no treatment) or 4% (transient puromycin treatment) (Figures 1C, middle and right panel, and 1D). Interestingly, we did not observe enhancement in donor incorporation following isolation of transiently transfected cells in this experiment, which may be due to the pac cassette presence on the AAVS1 donor-targeting construct and not on the gene-specific nuclease (pX330) plasmid. Therefore, direct comparisons with published methods where selection is performed for transfection of the nuclease containing plasmid (Ding et al., 2013a, 2013b) and CTS were not performed in this study. These data suggested that selecting for HDR-receptive cells via a selectable modification significantly enriched for cells bearing passenger modifications (precision edit events) at the site of interest. To benchmark the impact of CTS on HDR representation in a more quantitative manner and across multiple loci, we applied the workflow to a total of seven disease-associated variants across four different genes (CRYAB, BAG3, LMNA, and MTERF4) in two separate iPSC lines (hB53 hiPS6 and hB119 hiPS9) and analyzed editing outcomes via deep-sequencing. Following CTS, cells were pooled and deep-sequenced using the Illumina MiSeq platform. Average sequencing coverage following read trimming and quality filtering across all experiments was >150,000×, and the error rate was estimated to be less than 0.1%. We observed an average 3.7-fold (hB53 hiPS6) and 3.3-fold (hB119 hiPS9) increase in the total number of edits (HDR and NHEJ) across all loci with CTS compared with those without CTS (Table S2). Focusing on precision editing events and considering all seven variants, we observed an average 50-fold increase in HDR with CTS compared with those without CTS in both cell lines (Figures 2A–2C; Table S2). In support of our hypothesis that CTS enriches for HDR, we observed an overt shift in the balance between the two modes of repair such that the HDR/NHEJ ratio was enhanced on average 18-fold (hB53 hiPS6) and 27-fold (hB119 hiPS9) with CTS compared with those without CTS (Figures 2D–2F; Table S2). Considering all loci and both cell lines, the HDR rate following CTS was ∼14%, which corresponds to >1 in 10 clones bearing a precision edit. While CTS led to a gross increase in incorporation of the intended passenger modifications, deep-sequencing data revealed significant variation in locus targetability. For example, representation of the CRYAB:c.343delT variant was 22% (hB53 hiPS6) and 25% (hB119 hiPS9) following CTS (1% [hB53 hiPS6] and 3% [hB119 hiPS9] without CTS), whereas representation of the LMNA:c.1346G>T variant was 1% (hB53 hiPS6) and 4% (hB119 hiPS9) HDR following CTS (<0.05% in both cell lines without CTS). Notwithstanding, the average >100-fold increase in HDR at LMNA with CTS means that isolating a precision-edited clone is feasible (∼1 cell in 40 with CTS compared with ∼1 cell in ∼3,800 without, assuming heterozygosity) and suggests that loci which are inherently refractory to precision editing may be amenable via CTS. We also observed that the extent of ssODN incorporation was seemingly independent of its orientation relative to the sgRNA target strand (Table S3).