title: Efficient Gene Editing in Pluripotent Stem Cells by Bacterial Injection of Transcription Activator-Like Effector Nuclease Proteins.
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.
authors.affiliation: State Key Laboratory of Medical and Chemical Biology, College of Life Sciences, Nankai University, Tianjin, People's Republic of China; Department of Molecular Genetics and Microbiology and Department of Pathology, University of Florida, Gainesville, Florida, USA; Department of Biotechnology and Bioinformatics, Korea University, Sejong, Republic of Korea; Key Laboratory of Regenerative Biology, Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, People's Republic of China.
authors.forename: Jingyue
authors.initials: J
authors.lastname: Jia
authors.affiliation: State Key Laboratory of Medical and Chemical Biology, College of Life Sciences, Nankai University, Tianjin, People's Republic of China; Department of Molecular Genetics and Microbiology and Department of Pathology, University of Florida, Gainesville, Florida, USA; Department of Biotechnology and Bioinformatics, Korea University, Sejong, Republic of Korea; Key Laboratory of Regenerative Biology, Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, People's Republic of China.
authors.forename: Fang
authors.initials: F
authors.lastname: Bai
authors.affiliation: State Key Laboratory of Medical and Chemical Biology, College of Life Sciences, Nankai University, Tianjin, People's Republic of China; Department of Molecular Genetics and Microbiology and Department of Pathology, University of Florida, Gainesville, Florida, USA; Department of Biotechnology and Bioinformatics, Korea University, Sejong, Republic of Korea; Key Laboratory of Regenerative Biology, Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, People's Republic of China.
authors.forename: Yongxin
authors.initials: Y
authors.lastname: Jin
authors.affiliation: State Key Laboratory of Medical and Chemical Biology, College of Life Sciences, Nankai University, Tianjin, People's Republic of China; Department of Molecular Genetics and Microbiology and Department of Pathology, University of Florida, Gainesville, Florida, USA; Department of Biotechnology and Bioinformatics, Korea University, Sejong, Republic of Korea; Key Laboratory of Regenerative Biology, Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, People's Republic of China.
authors.forename: Katherine E
authors.initials: KE
authors.lastname: Santostefano
authors.affiliation: State Key Laboratory of Medical and Chemical Biology, College of Life Sciences, Nankai University, Tianjin, People's Republic of China; Department of Molecular Genetics and Microbiology and Department of Pathology, University of Florida, Gainesville, Florida, USA; Department of Biotechnology and Bioinformatics, Korea University, Sejong, Republic of Korea; Key Laboratory of Regenerative Biology, Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, People's Republic of China.
authors.forename: Un-Hwan
authors.initials: UH
authors.lastname: Ha
authors.affiliation: State Key Laboratory of Medical and Chemical Biology, College of Life Sciences, Nankai University, Tianjin, People's Republic of China; Department of Molecular Genetics and Microbiology and Department of Pathology, University of Florida, Gainesville, Florida, USA; Department of Biotechnology and Bioinformatics, Korea University, Sejong, Republic of Korea; Key Laboratory of Regenerative Biology, Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, People's Republic of China.
authors.forename: Donghai
authors.initials: D
authors.lastname: Wu
authors.affiliation: State Key Laboratory of Medical and Chemical Biology, College of Life Sciences, Nankai University, Tianjin, People's Republic of China; Department of Molecular Genetics and Microbiology and Department of Pathology, University of Florida, Gainesville, Florida, USA; Department of Biotechnology and Bioinformatics, Korea University, Sejong, Republic of Korea; Key Laboratory of Regenerative Biology, Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, People's Republic of China sjin@ufl.edu terada@pathology.ufl.edu wuweihui@nankai.edu.cn.
authors.forename: Weihui
authors.initials: W
authors.lastname: Wu
authors.affiliation: State Key Laboratory of Medical and Chemical Biology, College of Life Sciences, Nankai University, Tianjin, People's Republic of China; Department of Molecular Genetics and Microbiology and Department of Pathology, University of Florida, Gainesville, Florida, USA; Department of Biotechnology and Bioinformatics, Korea University, Sejong, Republic of Korea; Key Laboratory of Regenerative Biology, Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, People's Republic of China sjin@ufl.edu terada@pathology.ufl.edu wuweihui@nankai.edu.cn.
authors.forename: Naohiro
authors.initials: N
authors.lastname: Terada
authors.affiliation: State Key Laboratory of Medical and Chemical Biology, College of Life Sciences, Nankai University, Tianjin, People's Republic of China; Department of Molecular Genetics and Microbiology and Department of Pathology, University of Florida, Gainesville, Florida, USA; Department of Biotechnology and Bioinformatics, Korea University, Sejong, Republic of Korea; Key Laboratory of Regenerative Biology, Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, People's Republic of China sjin@ufl.edu terada@pathology.ufl.edu wuweihui@nankai.edu.cn.
authors.forename: Shouguang
authors.initials: S
authors.lastname: Jin
mesh_terms: D000818:Animals; D002454:Cell Differentiation / Q000235:genetics; D053595:Embryonic Stem Cells / Q000378:metabolism*; D004720:Endonucleases / Q000235:genetics*; D018014:Gene Transfer Techniques; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000378:metabolism*; D051379:Mice; D010957:Plasmids; D039904:Pluripotent Stem Cells / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000235:genetics; D015534:Trans-Activators / Q000235:genetics*; D015533:Transcriptional Activation / Q000235:genetics; D014162:Transfection
chemical_list: D015534:Trans-Activators; D004720:Endonucleases
keywords: Embryonic stem cell; Genome editing; Transcription activator-like effector nuclease; Type III secretion system; iPS cell