Homologous sequence targeting in eukaryotic cells
First Claim
1. A method for targeting and altering, by homologous recombination, a preselected target DNA sequence in a eukaryotic cell in vitro to make a targeted sequence modification, said method comprising:
- introducing into at least one eukaryotic cell at least one recombinase and at least two single-stranded targeting polynucleotides which are substantially complementary to each other, and which further comprise a homology clamp that substantially corresponds to or is substantially complementary to said preselected target DNA sequence and;
identifying a eukaryotic cell having a the at a preselected target DNA sequence.
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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 eukaryotic 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 eukaryotic cell, generating a DNA sequence-specific targeting of one or more chemical substituents in an intact nucleus of a metabolically active eukaryotic 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.
30 Citations
20 Claims
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1. A method for targeting and altering, by homologous recombination, a preselected target DNA sequence in a eukaryotic cell in vitro to make a targeted sequence modification, said method comprising:
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introducing into at least one eukaryotic cell at least one recombinase and at least two single-stranded targeting polynucleotides which are substantially complementary to each other, and which further comprise a homology clamp that substantially corresponds to or is substantially complementary to said preselected target DNA sequence and;
identifying a eukaryotic cell having a the at a preselected target DNA sequence. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)
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16. A method for generating a targeted sequence modification in a preselected endogenous target DNA sequence in a eukaryotic cell in vitro, comprising:
introducing into a eukaryotic cell, a recombinase having strand transfer activity and at least two single-stranded targeting polynucleotides which are substantially complementary to each other, and which further comprise a homology clamp that substantially corresponds to or is substantially complementary to said preselected endogenous target DNA sequence; and
identifying a eukaryotic cell having the targeted sequence modification at said preselected endogenous target DNA sequence.- View Dependent Claims (17, 18, 19)
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20. A method for targeting and altering, by homologous recombination, a preselected DNA sequence in a eukaryotic cell in vitro to make a targeted sequence modification, said method comprising introducing into at least one eukaryotic cell at least one recA recombinase and at least two single-stranded targeting polynucleotides which are substantially complementary to each other, and which further comprise a homology clamp that substantially corresponds to or is substantially complementary to a preselected target DNA sequence;
- and identifying a eukaryotic cell having the targetet DNA sequence modification at a preselected target DNA sequence.
Specification