title: Efficient, footprint-free human iPSC genome editing by consolidation of Cas9/CRISPR and piggyBac technologies.
abstract: Genome editing of human induced pluripotent stem cells (hiPSCs) offers unprecedented opportunities for in vitro disease modeling and personalized cell replacement therapy. The introduction of Cas9-directed genome editing has expanded adoption of this approach. However, marker-free genome editing using standard protocols remains inefficient, yielding desired targeted alleles at a rate of ∼1-5%. We developed a protocol based on a doxycycline-inducible Cas9 transgene carried on a piggyBac transposon to enable robust and highly efficient Cas9-directed genome editing, so that a parental line can be expeditiously engineered to harbor many separate mutations. Treatment with doxycycline and transfection with guide RNA (gRNA), donor DNA and piggyBac transposase resulted in efficient, targeted genome editing and concurrent scarless transgene excision. Using this approach, in 7 weeks it is possible to efficiently obtain genome-edited clones with minimal off-target mutagenesis and with indel mutation frequencies of 40-50% and homology-directed repair (HDR) frequencies of 10-20%.
authors.affiliation: Department of Cardiology, Boston, Massachusetts, USA.
authors.forename: Gang
authors.initials: G
authors.lastname: Wang
authors.affiliation: Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, Massachusetts, USA.|Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Luhan
authors.initials: L
authors.lastname: Yang
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Dennis
authors.initials: D
authors.lastname: Grishin
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Xavier
authors.initials: X
authors.lastname: Rios
authors.affiliation: Department of Cardiology, Boston, Massachusetts, USA.
authors.forename: Lillian Y
authors.initials: LY
authors.lastname: Ye
authors.affiliation: Department of Cardiology, Boston, Massachusetts, USA.
authors.forename: Yong
authors.initials: Y
authors.lastname: Hu
authors.affiliation: Department of Cardiology, Boston, Massachusetts, USA.
authors.forename: Kai
authors.initials: K
authors.lastname: Li
authors.affiliation: Department of Cardiology, Boston, Massachusetts, USA.
authors.forename: Donghui
authors.initials: D
authors.lastname: Zhang
authors.affiliation: Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, Massachusetts, USA.|Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: George M
authors.initials: GM
authors.lastname: Church
authors.affiliation: Department of Cardiology, Boston, Massachusetts, USA.|Harvard Stem Cell Institute, Harvard University, Cambridge, Massachusetts, USA.
authors.forename: William T
authors.initials: WT
authors.lastname: Pu
mesh_terms: D001483:Base Sequence; D064113:CRISPR-Cas Systems / Q000235:genetics*; D002460:Cell Line; D004318:Doxycycline / Q000494:pharmacology; D000072669:Gene Editing / Q000379:methods*; D015894:Genome, Human / Q000235:genetics*; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000187:drug effects / Q000378:metabolism*
chemical_list: D004318:Doxycycline