title: Integration-defective lentiviral vector mediates efficient gene editing through homology-directed repair in human embryonic stem cells.
abstract: Human embryonic stem cells (hESCs) are used as platforms for disease study, drug screening and cell-based therapy. To facilitate these applications, it is frequently necessary to genetically manipulate the hESC genome. Gene editing with engineered nucleases enables site-specific genetic modification of the human genome through homology-directed repair (HDR). However, the frequency of HDR remains low in hESCs. We combined efficient expression of engineered nucleases and integration-defective lentiviral vector (IDLV) transduction for donor template delivery to mediate HDR in hESC line WA09. This strategy led to highly efficient HDR with more than 80% of the selected WA09 clones harboring the transgene inserted at the targeted genomic locus. However, certain portions of the HDR clones contained the concatemeric IDLV genomic structure at the target site, probably resulted from recombination of the IDLV genomic input before HDR with the target. We found that the integrase protein of IDLV mediated the highly efficient HDR through the recruitment of a cellular protein, LEDGF/p75. This study demonstrates that IDLV-mediated HDR is a powerful and broadly applicable technology to carry out site-specific gene modification in hESCs.
authors.affiliation: Bone Marrow Transplantation Center, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang 310003, China.|Department of Diabetes and Metabolic Diseases Research, Beckman Research Institute, City of Hope National Medical Center, Duarte, CA 91010, USA.
authors.forename: Yebo
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Department of Pathology, School of Medicine, Hangzhou Normal University, Hangzhou, Zhejiang 310036, China.
authors.forename: Yingjia
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Department of Diabetes and Metabolic Diseases Research, Beckman Research Institute, City of Hope National Medical Center, Duarte, CA 91010, USA.
authors.forename: Tammy
authors.initials: T
authors.lastname: Chang
authors.affiliation: Bone Marrow Transplantation Center, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang 310003, China.
authors.forename: He
authors.initials: H
authors.lastname: Huang
authors.affiliation: Department of Diabetes and Metabolic Diseases Research, Beckman Research Institute, City of Hope National Medical Center, Duarte, CA 91010, USA.
authors.forename: Jiing-Kuan
authors.initials: JK
authors.lastname: Yee
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D002460:Cell Line; D044708:DNA, Concatenated / Q000235:genetics / Q000378:metabolism; D000072669:Gene Editing / Q000379:methods; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism*; D015894:Genome, Human; D000066449:Human Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D019426:Integrases / Q000235:genetics* / Q000378:metabolism; D016086:Lentivirus / Q000235:genetics* / Q000378:metabolism; D059767:Recombinational DNA Repair; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D044708:DNA, Concatenated; C504649:PSIP1 protein, human; D014157:Transcription Factors; D014764:Viral Proteins; D019426:Integrases