Methods for gene disruption and uses thereof
First Claim
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1. A method for disrupting a gene of an organism without introducing downstream effects, comprising:
- (a) providing a host cell of an organism, wherein the host cell is capable of homologous recombination;
(b) identifying an open reading frame for a gene of the organism;
(c) introducing into the host cell a targeting polynucleotide comprising (i) a molecular tag and (ii) flanking homology clamps for aligning the targeting polynucleotide in-frame with the open reading frame of the gene; and
(d) selecting host cells having an in-frame disruption of the gene.
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Abstract
The present invention relates to compositions and methods for in-frame disruption of a gene sequence by homologous recombination. The present invention may be used in certain embodiments to disrupt a gene without causing any downstream effects on non-target sequences. In certain embodiments, the inventive methods may be used to identify and/or characterize products encoded by essential genes, conditionally essential genes, and non-essential genes.
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Citations
30 Claims
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1. A method for disrupting a gene of an organism without introducing downstream effects, comprising:
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(a) providing a host cell of an organism, wherein the host cell is capable of homologous recombination;
(b) identifying an open reading frame for a gene of the organism;
(c) introducing into the host cell a targeting polynucleotide comprising (i) a molecular tag and (ii) flanking homology clamps for aligning the targeting polynucleotide in-frame with the open reading frame of the gene; and
(d) selecting host cells having an in-frame disruption of the gene. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 30)
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20. A method for characterizing the essentiality of a plurality of genes from an organism by in-frame gene disruption, comprising:
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(a) providing a host cell of an organism, wherein the host cell is capable of homologous recombination;
(b) identifying at least five open reading frames for at least five genes of the organism;
(c) introducing into the host cell a targeting polynucleotide for each of the genes comprising (i) a molecular tag and (ii) flanking homology clamps for aligning the targeting polynucleotides in-frame with the open reading frames of the genes;
(d) selecting host cells having in-frame disruptions of the genes; and
(e) determining whether the in-frame insertions of the targeting polynucleotides into the genome of the host cell produced a change in the phenotype of the host cell attributable to disruption of the genes in the absence of any downstream effects. - View Dependent Claims (21, 22, 23, 24, 25, 26, 27, 28, 29)
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Specification