title: Transcription activator-like effector nuclease (TALEN)-mediated CLYBL targeting enables enhanced transgene expression and one-step generation of dual reporter human induced pluripotent stem cell (iPSC) and neural stem cell (NSC) lines.
abstract: Targeted genome engineering to robustly express transgenes is an essential methodology for stem cell-based research and therapy. Although designer nucleases have been used to drastically enhance gene editing efficiency, targeted addition and stable expression of transgenes to date is limited at single gene/locus and mostly PPP1R12C/AAVS1 in human stem cells. Here we constructed transcription activator-like effector nucleases (TALENs) targeting the safe-harbor like gene CLYBL to mediate reporter gene integration at 38%-58% efficiency, and used both AAVS1-TALENs and CLYBL-TALENs to simultaneously knock-in multiple reporter genes at dual safe-harbor loci in human induced pluripotent stem cells (iPSCs) and neural stem cells (NSCs). The CLYBL-TALEN engineered cell lines maintained robust reporter expression during self-renewal and differentiation, and revealed that CLYBL targeting resulted in stronger transgene expression and less perturbation on local gene expression than PPP1R12C/AAVS1. TALEN-mediated CLYBL engineering provides improved transgene expression and options for multiple genetic modification in human stem cells.
authors.affiliation: NIH Center for Regenerative Medicine, Laboratory of Stem Cell Biology, National Institute of Arthritis, Musculoskeletal and Skin Diseases, Bethesda, Maryland, United States of America.
authors.forename: Trevor
authors.initials: T
authors.lastname: Cerbini
authors.affiliation: NIH Center for Regenerative Medicine, Laboratory of Stem Cell Biology, National Institute of Arthritis, Musculoskeletal and Skin Diseases, Bethesda, Maryland, United States of America.
authors.forename: Ray
authors.initials: R
authors.lastname: Funahashi
authors.affiliation: NIH Center for Regenerative Medicine, Laboratory of Stem Cell Biology, National Institute of Arthritis, Musculoskeletal and Skin Diseases, Bethesda, Maryland, United States of America.
authors.forename: Yongquan
authors.initials: Y
authors.lastname: Luo
authors.affiliation: Center for Molecular Medicine, Division of Intramural Research, National Heart, Lung, and Blood Institute, Bethesda, Maryland, United States of America.
authors.forename: Chengyu
authors.initials: C
authors.lastname: Liu
authors.affiliation: Stem Cell Unit, National Institute of Neurological Disorders and Stroke, Bethesda, Maryland, United States of America.
authors.forename: Kyeyoon
authors.initials: K
authors.lastname: Park
authors.affiliation: NIH Center for Regenerative Medicine, Laboratory of Stem Cell Biology, National Institute of Arthritis, Musculoskeletal and Skin Diseases, Bethesda, Maryland, United States of America.
authors.forename: Mahendra
authors.initials: M
authors.lastname: Rao
authors.affiliation: NIH Center for Regenerative Medicine, Laboratory of Stem Cell Biology, National Institute of Arthritis, Musculoskeletal and Skin Diseases, Bethesda, Maryland, United States of America.
authors.forename: Nasir
authors.initials: N
authors.lastname: Malik
authors.affiliation: NIH Center for Regenerative Medicine, Laboratory of Stem Cell Biology, National Institute of Arthritis, Musculoskeletal and Skin Diseases, Bethesda, Maryland, United States of America; Center for Molecular Medicine, Division of Intramural Research, National Heart, Lung, and Blood Institute, Bethesda, Maryland, United States of America.
authors.forename: Jizhong
authors.initials: J
authors.lastname: Zou
mesh_terms: D002460:Cell Line; D004720:Endonucleases / Q000235:genetics*; D018390:Gene Targeting / Q000379:methods*; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism; D058953:Neural Stem Cells / Q000166:cytology* / Q000378:metabolism; D019076:Transgenes / Q000235:genetics
chemical_list: D004720:Endonucleases