Methods and compositions for targeted gene modification
Inventors
Essner, Jeffrey J. • Liao, Hsin-Kai • Fahrenkrug, Scott C.
Assignees
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Publication Number
US-9074224-B2
Publication Date
2015-07-07
Expiration Date
Abstract
Disclosed herein are methods and compositions for gene targeting utilizing fusion molecules comprising a recombinase domain and a sequence-specific DNA-binding domain.
Core Innovation
The disclosed approach places an exogenous nucleic acid into DNA of a cell by introducing an exogenous nucleic acid together with a proteinaceous fusion molecule and a single strand DNA probe complementary to a target site in DNA of the cell. The probe and the recombinase domain are complexed as a nucleoprotein filament at the time of introduction. The exogenous nucleic acid is chosen for incorporation into the DNA of the cell and is designed to be free of specific binding to the proteinaceous fusion molecule.
The proteinaceous fusion molecule comprises a recombinase domain, a protein-protein interaction domain, and a nuclear localization signal domain. The method incorporates the exogenous nucleic acid into the DNA of the cell and expresses the incorporated nucleic acid by the cell. The nucleoprotein filament formation is used together with the complementary cssDNA targeting to achieve DNA-level placement at or near a target site.
A key constraint is that the method is performed without introducing nucleic acids into the cell that have specific binding to the protein-protein interaction domain. This constraint is used alongside the targeted filament pairing and functional requirements for the sequence-specific DNA-binding activity as exemplified by Gal4 to maintain specificity of the genome modification process.
Claims Coverage
The independent claim is directed to a method for placing an exogenous nucleic acid into DNA of a cell using a recombinase/protein-protein interaction/nuclear localization fusion molecule complexed with complementary cssDNA as a nucleoprotein filament at introduction time, together with constraints on the exogenous nucleic acid and on excluding nucleic acids that bind the protein-protein interaction domain. The dependent claims refine the independent claim with additional structural selections, DNA format constraints, and defined efficiency or relationship constraints.
Fusion-mediated nucleoprotein filament with complementary cssDNA targeting
A method comprising introducing a proteinaceous fusion molecule that comprises a recombinase domain, a protein-protein interaction domain, and a nuclear localization signal domain, and introducing a single strand DNA probe complementary to a target site in DNA of the cell (cssDNA), wherein the probe and the recombinase domain are complexed as a nucleoprotein filament at the time of the introduction into the cell.
Exogenous nucleic acid incorporation and expression without specific binding to fusion
The exogenous nucleic acid sequence is chosen for incorporation into the DNA of the cell and designed to be free of specific binding to the proteinaceous fusion molecule, wherein the exogenous nucleic acid is incorporated into the DNA of the cell and expressed by the cell.
Avoidance of nucleic acids binding the protein-protein interaction domain
The method is performed without introducing nucleic acids into the cell that have specific binding to the protein-protein interaction domain.
Across the independent-claim framework, the key inventive elements are targeted nucleoprotein filament formation using a fusion molecule containing a recombinase domain plus protein-protein interaction and nuclear localization domains, incorporation and expression of an exogenous nucleic acid that does not specifically bind the fusion molecule, and performing the method while excluding nucleic acids that specifically bind the protein-protein interaction domain.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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