Methods for gene disruption and uses thereof
OwnerDonald Awrey, Jack Greenblattinventors
Life of this patent
Estimating the term…
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.
Claims30
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.
A method for characterizing the essentiality of a plurality of genes from an organism by in-frame gene disruption, comprising:
- (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.
Dependencies are inferred from the claim text.