Pretesting of TALENs and mutational spectra in 293 T cells and hPSCs. A: Illustration of GoldenGate TALEN assembly by Cermak et al.  and cloning into new expression vectors pTAL7A and pTALB. Note that the number of RVDs is not limited to 18 but flexible. B: Schematic of negative selection of HPRT1 knock-out cells by 6-thioguanine (6-TG). 6-TG results in death of cells with functional HPRT1. C: Cel-1 assay following transfection with three TALEN pairs (#1 to #3). Arrows indicate expected fragment sizes following Cel-1 digest of reannealed HPRT1 genomic PCRs (gPCR) as a result of error-prone NHEJ. Control plasmid (GFP) transfected cells served as negative control. Percentage of non-homologous end-joining (NHEJ) is indicated below each lane. D: Surviving 293 T cells following TALEN transfection and 6-TG selection of HPRT1 mutants. E: TALEN-induced mutation spectrum. Underlined wild-type sequence indicates binding region of TALEN pair #2. Mutant sequences shown are individual gPCR clones from bulk 6-TG selected cultures following transfection of TALEN pair #2. F: Pretesting of TALEN pairs #1 to #3 in HuES6 hESCs. Note that mutation efficiencies are somewhat decreased compared to 293 T cells in part B. G: Bulk hESC cultures following TALEN #2 transfection and 6-TG selection. H: TALEN-induced mutation spectrum in hESCs. All transfected cells were enriched by transient puromycin/blasticidin administration.