The development of human embryonic stem cells (ESCs) and induced pluripotent 
stem cells (iPSCs) facilitates in vitro studies of human disease mechanisms, 
speeds up the process of drug screening, and raises the feasibility of using 
cell replacement therapy in clinics. However, the study of genotype-phenotype 
relationships in ESCs or iPSCs is hampered by the low efficiency of 
site-specific gene editing. Transcription activator-like effector nucleases 
(TALENs) spurred interest due to the ease of assembly, high efficiency and 
faithful gene targeting. In this study, we optimized the TALEN design to 
maximize its genomic cutting efficiency. We showed that using optimized TALENs 
in conjunction with single-strand oligodeoxynucleotide (ssODN) allowed efficient 
gene editing in human cells. Gene mutations and gene deletions for up to 7.8 kb 
can be accomplished at high efficiencies. We established human tumor cell lines 
and H9 ESC lines with homozygous deletion of the microRNA-21 (miR-21) gene and 
miR-9-2 gene. These cell lines provide a robust platform to dissect the roles 
these genes play during cell differentiation and tumorigenesis. We also observed 
that the endogenous homologous chromosome can serve as a donor template for gene 
editing. Overall, our studies demonstrate the versatility of using ssODN and 
TALEN to establish genetically modified cells for research and therapeutic 
application.