title: A robust TALENs system for highly efficient mammalian genome editing.
abstract: Recently, transcription activator-like effector nucleases (TALENs) have emerged as a highly effective tool for genomic editing. A pair of TALENs binds to two DNA recognition sites separated by a spacer sequence, and the dimerized FokI nucleases at the C terminal then cleave DNA in the spacer. Because of its modular design and capacity to precisely target almost any desired genomic locus, TALEN is a technology that can revolutionize the entire biomedical research field. Currently, for genomic editing in cultured cells, two plasmids encoding a pair of TALENs are co-transfected, followed by limited dilution to isolate cell colonies with the intended genomic manipulation. However, uncertain transfection efficiency becomes a bottleneck, especially in hard-to-transfect cells, reducing the overall efficiency of genome editing. We have developed a robust TALENs system in which each TALEN plasmid also encodes a fluorescence protein. Thus, cells transfected with both TALEN plasmids, a prerequisite for genomic editing, can be isolated by fluorescence-activated cell sorting. Our improved TALENs system can be applied to all cultured cells to achieve highly efficient genomic editing. Furthermore, an optimized procedure for genomic editing using TALENs is also presented. We expect our system to be widely adopted by the scientific community.
authors.affiliation: 1] Magee-Womens Research Institute, Department of Obstetrics, Gynecology and Reproductive Sciences, University of Pittsburgh School of Medicine, Pittsburgh, PA, 15213, USA [2] Department of Internal Medical Oncology, The Third Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang Province 150081, China [3].
authors.forename: Yuanxi
authors.initials: Y
authors.lastname: Feng
authors.affiliation: 1] Magee-Womens Research Institute, Department of Obstetrics, Gynecology and Reproductive Sciences, University of Pittsburgh School of Medicine, Pittsburgh, PA, 15213, USA [2] Department of Radiation Oncology, The Third Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang Province 150081, China [3].
authors.forename: Siliang
authors.initials: S
authors.lastname: Zhang
authors.affiliation: 1] Magee-Womens Research Institute, Department of Obstetrics, Gynecology and Reproductive Sciences, University of Pittsburgh School of Medicine, Pittsburgh, PA, 15213, USA [2] Women's Cancer Research Center, University of Pittsburgh Cancer Institute, Pittsburgh, PA, 15213, USA.
authors.forename: Xin
authors.initials: X
authors.lastname: Huang
mesh_terms: D000595:Amino Acid Sequence; D015153:Blotting, Western; D045744:Cell Line, Tumor; D005456:Fluorescent Dyes / Q000737:chemistry; D016678:Genome; D006801:Humans; D008845:Microinjections; D008969:Molecular Sequence Data; D017393:RNA Editing; D012333:RNA, Messenger / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012260:Ribonucleases / Q000378:metabolism*; D015534:Trans-Activators / Q000378:metabolism*; D014162:Transfection
chemical_list: D005456:Fluorescent Dyes; D012333:RNA, Messenger; D015534:Trans-Activators; D012260:Ribonucleases