Methods of enhancing chromosomal homologous recombination
Described herein are methods of enhancing chromosomal homologous recombination to stimulate a loss of heterozygosity at a gene locus of interest in a living cell. These methods are driven by an enhancer component and a target-specific endonuclease component and proceed through a mechanism whereby: exogenous donor DNA that is homologous to the gene locus of interest is not introduced into the living cell; the desired allele of the gene locus of interest remains uncleaved; and the undesired allele is either uncleaved, cleaved at a single location, or cleaved at multiple locations. These methods have numerous applications, including the repair of risk alleles for disease prevention, the correction of heterozygous mutations in dividing cells, the design of cancer therapeutics, and the design of novel gene-drive strategies.
1. A method of stimulating a loss of heterozygosity at a gene locus of interest in a living cell comprising:
identifying a gene locus of interest in a living cell, wherein the gene locus of interest comprises a desired allele and an undesired allele; and
introducing an enzymatic unit into the living cell, wherein the enzymatic unit enhances interchromosomal homologous recombination between the desired allele and the undesired allele of the gene locus of interest and wherein the enzymatic unit comprises:
an enhancer component comprising Rad51; and
a target-specific endonuclease component, wherein the target-specific endonuclease component: (I) provides the enzymatic unit with target specificity; and (ii) has or alternatively lacks endonuclease activity.
2. The method of claim 1 , wherein the enzymatic unit cleaves the undesired allele of the gene locus of interest and wherein:
a. the undesired allele of the gene locus of interest is cleaved only once;
b. the desired allele of the gene locus of interest remains uncleaved;
c. the interchromosomal homologous recombination generates homozygosity at the gene locus of interest, wherein the gene of interest comprises two desired alleles after interchromosomal homologous recombination; and
d. exogenous donor DNA is not introduced into the living cell, wherein the exogenous donor DNA comprises a polynucleic acid sequence that is homologous to the gene locus of interest.
3. The method of claim 2 , wherein:
a. the enzymatic unit cleaves the undesired allele of the gene locus of interest through the introduction of a single-strand break; or
b. the enzymatic unit cleaves the undesired allele of the gene locus of interest through the introduction of a double-strand break.
4. The method of claim 1 , wherein the enzymatic unit lacks the ability to cleave DNA; and wherein:
a. the interchromosomal homologous recombination generates homozygosity at the gene locus of interest, wherein the gene of interest comprises two desired alleles after interchromosomal homologous recombination; and
b. exogenous donor DNA is not introduced into the living cell, wherein the exogenous donor DNA comprises a poly nucleic acid sequence that is homologous to the gene locus of interest.
5. The method of claim 4 , wherein the target-specific endonuclease component specifically binds to the undesired allele of the gene locus of interest.
6. The method of claim 1 , wherein;
a. the enhancer component comprises a poly nucleic acid that encodes for a poly peptide sequence that comprises the polypeptide sequence of Rad51; or
b. the enhancer component comprises a polypeptide that comprises the polypeptide sequence of Rad51.
7. The method of claim 1 , wherein the enhancer component further comprises a small molecule that enhances homologous directed repair.
8. The method of claim 7 , wherein the small molecule that enhances homologous directed repair is RS-1.
9. The method of claim 1 , wherein the target-specific endonuclease component comprises:
a. a polynucleic acid that encodes for a polypeptide sequence that comprises the polypeptide sequence of a target-specific endonuclease; or
b. a polypeptide that comprises the polypeptide sequence of a target-specific endonuclease.
10. The method of claim 9 , wherein the target-specific endonuclease is a RNA-dependent endonuclease.
11. The method of claim 10 , wherein;
a. the RNA-dependent endonuclease is a CRISPR/Cas protein;
b. the enzymatic unit further comprises a crRNA, a tracrRNA, and/or a sgRNA; or
c. a combination thereof.
12. The method of claim 11 , wherein the CRISPR/Cas protein is a mutated CRISPR/Cas protein.
13. The method of claim 12 , wherein the mutated CRISPR/Cas protein comprises a mutated nuclease domain and wherein the mutated CRISPR/Cas protein generates single-strand breaks.
14. The method of claim 9 , wherein the target-specific endonuclease is a RNA-independent endonuclease.
15. The method of claim 14 , wherein the RNA-independent endonuclease is selected from the group consisting of a meganuclease, a zinc-finger nuclease, a transcription activator-like effector nuclease, and a restriction enzyme, optionally wherein the restriction enzyme is a site-specific DNA-nicking enzyme.
16. The method of claim 1 , wherein the loss of heterozygosity is enhanced by at least 20% relative to the loss of heterozygosity in the absence of the at least one enhancer component.
17. The method of claim 1 , wherein the enhancer component stimulates essentially error-free interchromosomal homologous recombination.
18. The method of claim 1 , wherein the living cell is;
a. a cell of a multicellular organism, wherein the multicellular organism is administered a composition comprising: the at least one enhancer component; the target-specific endonuclease component; and optionally a cellular delivery component; or
b. a unicellular organism.
19. The method of claim 18 , wherein the multicellular organism is a human.
20. The method of claim 19 , wherein the human is a patient suffering from a disease, wherein the disease is caused by heterozygosity a gene locus of interest.
21. The method of claim 18 wherein the unicellular organism is a bacteria.
22. The method of claim 1 , wherein the living cell is a cell of a genetically engineered transgenic organism and wherein the gene locus of interest comprises a transgenic gene locus, wherein the desired allele of the transgenic gene locus is a transgenic payload gene and the undesired allele of the transgenic gene locus is an endogenous sequence.
23. A therapeutic composition for use in a method of treating a medical condition caused by heterozygosity at an allele of a gene of interest, the composition comprising:
an enhancer component comprising Rad51;
a target-specific endonuclease component wherein the target-specific endonuclease component has or alternatively lacks endonuclease activity; and
optionally a cellular delivery component.
24. The therapeutic composition of claim 23 , wherein the medical condition is:
a. an autosomal dominant disorder; or
b. caused by codominance or incomplete dominance.