title: Multi-kilobase homozygous targeted gene replacement in human induced pluripotent stem cells.
abstract: Sequence-specific nucleases such as TALEN and the CRISPR/Cas9 system have so far been used to disrupt, correct or insert transgenes at precise locations in mammalian genomes. We demonstrate efficient 'knock-in' targeted replacement of multi-kilobase genes in human induced pluripotent stem cells (iPSC). Using a model system replacing endogenous human genes with their mouse counterpart, we performed a comprehensive study of targeting vector design parameters for homologous recombination. A 2.7 kilobase (kb) homozygous gene replacement was achieved in up to 11% of iPSC without selection. The optimal homology arm length was around 2 kb, with homology length being especially critical on the arm not adjacent to the cut site. Homologous sequence inside the cut sites was detrimental to targeting efficiency, consistent with a synthesis-dependent strand annealing (SDSA) mechanism. Using two nuclease sites, we observed a high degree of gene excisions and inversions, which sometimes occurred more frequently than indel mutations. While homozygous deletions of 86 kb were achieved with up to 8% frequency, deletion frequencies were not solely a function of nuclease activity and deletion size. Our results analyzing the optimal parameters for targeting vector design will inform future gene targeting efforts involving multi-kilobase gene segments, particularly in human iPSC.
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, MA 02115, USA sbyrne@genetics.med.harvard.edu.
authors.forename: Susan M
authors.initials: SM
authors.lastname: Byrne
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Luis
authors.initials: L
authors.lastname: Ortiz
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Prashant
authors.initials: P
authors.lastname: Mali
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: John
authors.initials: J
authors.lastname: Aach
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, MA 02115, USA Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA 02115, USA gchurch@genetics.med.harvard.edu.
authors.forename: George M
authors.initials: GM
authors.lastname: Church
mesh_terms: D001483:Base Sequence; D002469:Cell Separation; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D017931:DNA Primers; D005434:Flow Cytometry; D017353:Gene Deletion; D006720:Homozygote; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000378:metabolism*; D010957:Plasmids; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
chemical_list: D017931:DNA Primers