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Nucleic Acids Res. 2015 Feb 18;43(3):e21. doi: 10.1093/nar/gku1246. Epub 2014 Nov 20.

Multi-kilobase homozygous targeted gene replacement in human induced pluripotent stem cells.

Author information

1
Department of Genetics, Harvard Medical School, Boston, MA 02115, USA sbyrne@genetics.med.harvard.edu.
2
Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
3
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.

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.

PMID:
25414332
PMCID:
PMC4330342
DOI:
10.1093/nar/gku1246
[Indexed for MEDLINE]
Free PMC Article

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