Sequence alterations using homologous recombination
First Claim
1. A method for targeting and altering, by homologous recombination, a pre-selected target nucleic acid sequence in an extrachromosomal sequence of a procaryotic cell, said method comprising:
- a) adding to said extrachromosomal sequence at least one recombinase and at least two single-stranded targeting polynucleotides each of which are substantially complementary to each other and comprise a homology clamp that substantially corresponds to or is substantially complementary to a preselected target nucleic acid sequence to form an altered extrachromosomal sequence; and
b) introducing said altered sequence into a procaryotic cell.
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Abstract
The invention relates to methods for targeting an exogenous polynucleotide or exogenous complementary polynucleotide pair to a predetermined endogenous DNA target sequence in a target cell by homologous pairing, particularly for altering an endogenous DNA sequence, such as a chromosomal DNA sequence, typically by targeted homologous recombination. In certain embodiments, the invention relates to methods for targeting an exogenous polynucleotide having a linked chemical substituent to a predetermined endogenous DNA sequence in a metabolically active target cell, generating a DNA sequence-specific targeting of one or more chemical substituents in an intact nucleus of a metabolically active target cell, generally for purposes of altering a predetermined endogenous DNA sequence in the cell. The invention also relates to compositions that contain exogenous targeting polynucleotides, complementary pairs of exogenous targeting polynucleotides, chemical substituents of such polynucleotides, and recombinase proteins used in the methods of the invention.
52 Citations
39 Claims
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1. A method for targeting and altering, by homologous recombination, a pre-selected target nucleic acid sequence in an extrachromosomal sequence of a procaryotic cell, said method comprising:
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a) adding to said extrachromosomal sequence at least one recombinase and at least two single-stranded targeting polynucleotides each of which are substantially complementary to each other and comprise a homology clamp that substantially corresponds to or is substantially complementary to a preselected target nucleic acid sequence to form an altered extrachromosomal sequence; and
b) introducing said altered sequence into a procaryotic cell. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25)
microinjection, electroporation, laser poration, biolistics, and liposome mediated transfection.
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23. A method according to claim 15, wherein said chemical substituent is covalently attached to said at least one targeting polynucleotide.
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24. A method according to claim 16, wherein said recA protein is E. coli recA protein.
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25. A method according to claim 19, wherein the cell-uptake component comprises a protein-lipid complex.
- 26. A method of generating a library of variant nucleic acid sequences of a pre-selected target nucleic acid sequence, said method comprising adding to said pre-selected target nucleic acid sequence at least one recombinase and a plurality of pairs of single-stranded targeting polynucleotides which are substantially complementary to each other and each comprising a homology clamp that substantially corresponds to or is substantially complementary to a preselected target nucleic acid sequence, said plurality of pairs comprising a library of mismatches between said targeting polynucleotide and said target nucleic acid sequence, to form a library of variant nucleic acid sequences.
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29. A method of generating a cellular library comprising variant nucleic acid sequences of a pre-selected target nucleic acid sequence, said method comprising introducing into a population of target cells at least one recombinase and a plurality of pairs of single-stranded targeting polynucleotides which are substantially complementary to each other and each comprising a homology clamp that substantially corresponds to or is substantially complementary to a preselected target nucleic acid sequence, said plurality of pairs comprising a library of mismatches between said targeting polynucleotide and said target nucleic acid sequence, to form said cellular library comprising variant nucleic acid sequences.
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31. A method of generating a cellular library comprising variant nucleic acid sequences of a pre-selected target nucleic acid sequence, said method comprising:
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a) adding to said pre-selected nucleic acid sequence at least one recombinase and a plurality of pairs of single-stranded targeting polynucleotides which are substantially complementary to each other and each comprising a homology clamp that substantially corresponds to or is substantially complementary to said preselected target nucleic acid sequence, said plurality of pairs comprising a library of mismatches between said targeting polynucleotide and said pre-selected target nucleic acid sequence, to form a plurality of altered sequences; and
b) introducing said altered sequences into a population of target cells to form said cellular library comprising variant nucleic acid sequences. - View Dependent Claims (32, 33)
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- 35. A composition comprising at least one eucaryotic recombinase and a variant library comprising a plurality of pairs of single stranded targeting polynucleotides which are substantially complementary to each other and each comprising a homology clamp that substantially corresponds to or is substantially complementary to a preselected target nucleic acid sequence, said plurality of pairs comprising a library of mismatches between said targeting polynucleotide and said target nucleic acid sequence.
Specification