Sequence alterations using homologous recombination
First Claim
1. A method for targeting and modifying, 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 extrachromosmal 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 a modified extrachromosomal sequence, wherein the target nucleic acid sequence is modified;
b) removing said recombinase; and
c) introducing said modified extrachromosomal sequence into a procaryotic cell.
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Accused Products
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.
74 Citations
40 Claims
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1. A method for targeting and modifying, 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 extrachromosmal 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 a modified extrachromosomal sequence, wherein the target nucleic acid sequence is modified; b) removing said recombinase; and c) introducing said modified extrachromosomal 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, 26)
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- 27. A method of generating a pool of variant nucleic acid sequences of a pre-selected target nucleic acid sequence in an extrachromosomal sequence, said method comprising adding to said extrachromosomal 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 altered extrachromosomal 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 in an extrachromosomal sequence of a target cell, said method comprising:
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a) adding to said extrachromosomal 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 plurality of altered extrachromosomal sequences; b) removing said recombinase; and c) introducing said altered sequences into a population of target cells to form said library of variant nucleic acid sequences. - View Dependent Claims (32, 33)
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- 35. A composition comprising at least one 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