title: Gene editing of human embryonic stem cells via an engineered baculoviral vector carrying zinc-finger nucleases.
abstract: Human embryonic stem (hES) cells are renewable cell sources that have potential applications in regenerative medicine. The development of technologies to produce permanent and site-specific genome modifications is in demand to achieve future medical implementation of hES cells. We report herein that a baculoviral vector (BV) system carrying zinc-finger nucleases (ZFNs) can successfully modify the hES cell genome. BV-mediated transient expression of ZFNs specifically disrupted the CCR5 locus in transduced cells and the modified cells exhibited resistance to HIV-1 transduction. To convert the BV to a gene targeting vector, a DNA donor template and ZFNs were incorporated into the vector. These hybrid vectors yielded permanent site-specific gene addition in both immortalized human cell lines (10%) and hES cells (5%). Modified hES cells were both karyotypically normal and pluripotent. These results suggest that this baculoviral delivery system can be engineered for site-specific genetic manipulation in hES cells.
authors.affiliation: Mork Family Department of Chemical Engineering and Materials Science, University of Southern California, Los Angeles, California 90089, USA.
authors.forename: Yuning
authors.initials: Y
authors.lastname: Lei
authors.forename: Chi-Lin
authors.initials: CL
authors.lastname: Lee
authors.forename: Kye-Il
authors.initials: KI
authors.lastname: Joo
authors.forename: Jonathan
authors.initials: J
authors.lastname: Zarzar
authors.forename: Yarong
authors.initials: Y
authors.lastname: Liu
authors.forename: Bingbing
authors.initials: B
authors.lastname: Dai
authors.forename: Victoria
authors.initials: V
authors.lastname: Fox
authors.forename: Pin
authors.initials: P
authors.lastname: Wang
mesh_terms: D002460:Cell Line; D003851:Deoxyribonucleases / Q000235:genetics* / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000378:metabolism*; D005434:Flow Cytometry; D018390:Gene Targeting; D005822:Genetic Vectors; D015497:HIV-1 / Q000235:genetics; D006801:Humans; D009154:Mutation; D017924:Nucleopolyhedroviruses / Q000235:genetics*; D019713:Receptors, CCR5 / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014161:Transduction, Genetic; D019076:Transgenes; D016335:Zinc Fingers
chemical_list: D019713:Receptors, CCR5; D003851:Deoxyribonucleases