Efficient Gene Editing in Pluripotent Stem Cells by Bacterial Injection of Transcription Activator-Like Effector Nuclease Proteins
- Authors
- Jia, Jingyue; Bai, Fang; Jin, Yongxin; Santostefano, Katherine E.; Ha, Un-Hwan; Wu, Donghai; Wu, Weihui; Terada, Naohiro; Jin, Shouguang
- Issue Date
- 8월-2015
- Publisher
- WILEY
- Keywords
- Transcription activator-like effector nuclease; Type III secretion system; Embryonic stem cell; iPS cell; Genome editing
- Citation
- STEM CELLS TRANSLATIONAL MEDICINE, v.4, no.8, pp.913 - 926
- Indexed
- SCIE
SCOPUS
- Journal Title
- STEM CELLS TRANSLATIONAL MEDICINE
- Volume
- 4
- Number
- 8
- Start Page
- 913
- End Page
- 926
- URI
- https://scholar.korea.ac.kr/handle/2021.sw.korea/92911
- DOI
- 10.5966/sctm.2015-0030
- ISSN
- 2157-6564
- Abstract
- The type III secretion system (T3SS) of Pseudomonas aeruginosa is a powerful tool for direct protein delivery into mammalian cells and has successfully been used to deliver various exogenous proteins into mammalian cells. In the present study, transcription activator-like effector nuclease (TALEN) proteins have been efficiently delivered using the P. aeruginosa T3SS into mouse embryonic stem cells (mESCs), human ESCs (hESCs), and human induced pluripotent stem cells (hiPSCs) for genome editing. This bacterial delivery system offers an alternative method of TALEN delivery that is highly efficient in cleavage of the chromosomal target and presumably safer by avoiding plasmid DNA introduction. We combined the method of bacterial T3SS-mediated TALEN protein injection and transfection of an oligonucleotide template to effectively generate precise genetic modifications in the stem cells. Initially, we efficiently edited a single-base in the gfp gene of a mESC line to silence green fluorescent protein (GFP) production. The resulting GFP-negative mESC was cloned from a single cell and subsequently mutated back to a GFP-positive mESC line. Using the same approach, the gfp gene was also effectively knocked out in hESCs. In addition, a defined single-base edition was effectively introduced into the X-chromosome-linked HPRT1 gene in hiPSCs, generating an in vitro model of Lesch-Nyhan syndrome. T3SS-mediated TALEN protein delivery provides a highly efficient alternative for introducing precise gene editing within pluripotent stem cells for the purpose of disease genotype-phenotype relationship studies and cellular replacement therapies.
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Collections - Graduate School > Department of Biotechnology and Bioinformatics > 1. Journal Articles
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