title: Optimization of scarless human stem cell genome editing.
abstract: Efficient strategies for precise genome editing in human-induced pluripotent cells (hiPSCs) will enable sophisticated genome engineering for research and clinical purposes. The development of programmable sequence-specific nucleases such as Transcription Activator-Like Effectors Nucleases (TALENs) and Cas9-gRNA allows genetic modifications to be made more efficiently at targeted sites of interest. However, many opportunities remain to optimize these tools and to enlarge their spheres of application. We present several improvements: First, we developed functional re-coded TALEs (reTALEs), which not only enable simple one-pot TALE synthesis but also allow TALE-based applications to be performed using lentiviral vectors. We then compared genome-editing efficiencies in hiPSCs mediated by 15 pairs of reTALENs and Cas9-gRNA targeting CCR5 and optimized ssODN design in conjunction with both methods for introducing specific mutations. We found Cas9-gRNA achieved 7-8× higher non-homologous end joining efficiencies (3%) than reTALENs (0.4%) and moderately superior homology-directed repair efficiencies (1.0 versus 0.6%) when combined with ssODN donors in hiPSCs. Using the optimal design, we demonstrated a streamlined process to generated seamlessly genome corrected hiPSCs within 3 weeks.
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, 02115 MA, USA, Biological and Biomedical Sciences Program, Harvard Medical School, Boston, 02115 MA, USA, Children's Hospital, Boston, 02115 MA, USA, Chemistry and Chemical Biology program, Harvard, 02138 Cambridge, MA, USA and Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, 02138 MA, USA.
authors.forename: Luhan
authors.initials: L
authors.lastname: Yang
authors.forename: Marc
authors.initials: M
authors.lastname: Guell
authors.forename: Susan
authors.initials: S
authors.lastname: Byrne
authors.forename: Joyce L
authors.initials: JL
authors.lastname: Yang
authors.forename: Alejandro
authors.initials: A
authors.lastname: De Los Angeles
authors.forename: Prashant
authors.initials: P
authors.lastname: Mali
authors.forename: John
authors.initials: J
authors.lastname: Aach
authors.forename: Caroline
authors.initials: C
authors.lastname: Kim-Kiselak
authors.forename: Adrian W
authors.initials: AW
authors.lastname: Briggs
authors.forename: Xavier
authors.initials: X
authors.lastname: Rios
authors.forename: Po-Yi
authors.initials: PY
authors.lastname: Huang
authors.forename: George
authors.initials: G
authors.lastname: Daley
authors.forename: George
authors.initials: G
authors.lastname: Church
mesh_terms: D002460:Cell Line; D002469:Cell Separation; D003851:Deoxyribonucleases / Q000737:chemistry / Q000378:metabolism*; D056426:Genetic Loci; D015894:Genome, Human; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides; D059767:Recombinational DNA Repair; D052416:Targeted Gene Repair / Q000379:methods*; D058727:RNA, Small Untranslated
chemical_list: D009838:Oligodeoxyribonucleotides; D003851:Deoxyribonucleases; D058727:RNA, Small Untranslated
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