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- Title
Precise Gene Modification Mediated by TALEN and Single-Stranded Oligodeoxynucleotides in Human Cells.
- Authors
Wang, Xiaoling; Wang, Yingjia; Huang, He; Chen, Buyuan; Chen, Xinji; Hu, Jianda; Chang, Tammy; Lin, Ren-Jang; Yee, Jiing-Kuan
- Abstract
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.
- Subjects
SINGLE-stranded DNA; HUMAN embryonic stem cells; INDUCED pluripotent stem cells; DRUG use testing; STEM cell treatment; GENE targeting; HUMAN cell culture; TAL effectors; DNA restriction enzymes
- Publication
PLoS ONE, 2014, Vol 9, Issue 4, p1
- ISSN
1932-6203
- Publication type
Article
- DOI
10.1371/journal.pone.0093575