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Methods and compositions for nuclease-mediated targeted integration of transgenes

  • US 10,174,331 B2
  • Filed: 05/07/2013
  • Issued: 01/08/2019
  • Est. Priority Date: 05/07/2012
  • Status: Active Grant
First Claim
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1. A method of integrating a transgene into an endogenous locus of a cell, the method comprising;

  • introducing one or more nucleases into the cell, the cell comprising a circular double-stranded plasmid donor, the double-stranded plasmid donor comprising;

    a transgene; and

    first and second sequences flanking the transgene, the first and second sequences flanking the transgene comprising paired target sites for one or more nucleases, the target sites flanking a spacer sequence, wherein at least one spacer sequence differs from the spacer sequence present in the genome of the cell, wherein the double-stranded plasmid does not comprise homology arms and further wherein the nucleases cleave the double-stranded plasmid in the sequences flanking the transgene and cleave the endogenous locus such that the transgene is integrated into the endogenous locus via homology-independent mechanisms.

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