Figure 1

Pretesting of TALENs and mutational spectra in 293 T cells and hPSCs. A: Illustration of GoldenGate TALEN assembly by Cermak et al. [13] 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.