(A) Customized TALENs are designed for a specific target site based on several criteria (see Section 3.1) and cloned using a library of tetramer and trimer domains into two pTALEN expression plasmids. (B) TALEN expression plasmids with or without ssODN or recombination plasmid DNA are introduced into hPSCs by electroporation. (C) 48 hours after electroporation, GFP positive, RFP positive, or double positive TALEN expressing hPSCs, are purified by FACS. (D) FACS isolated hPSCs are plated at a clonal density on 10 cm plates and clones are allowed to grow for 10-12 days. (E) hPSC clones are manually picked into 96-well plates – 1 hPSC clone per well. (F) 3–4 days after picking, each 96-well plate is passaged two plates, one Geltrex coated plate in which the cells will be grown to confluence for genomic DNA preparation and PCR screening (H) and one uncoated plate, which contains a frozen stock of each clonal cell line and is at –80 degrees Celsius (G). The entire process takes approximately 1 to 2 months depending on how quickly step A is completed.