Compositions and methods for increasing homology-directed repair
AssigneeThe Regents of the University of California
InventorsJennifer A. Doudna, Enrique Lin-Shiao, Rachel Lew
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Abstract
The present disclosure provides compositions comprising a gene-editing polypeptide, a single-stranded donor DNA, and one or more staple oligonucleotides. The present disclosure provides compositions comprising a DNA nanostructure and a gene-editing polypeptide. The present disclosure provides gene editing methods using the compositions. The present disclosure provides methods of using the compositions to produce a genetically modified cell. The present disclosure provides kits useful for carrying out gene editing.
Claims48
A composition comprising:
- a) a gene-editing polypeptide;
- b) a single-stranded donor DNA comprising:
- i) a first homology arm at or near the 5′ end of the donor DNA, wherein the first homology arm comprises a nucleotide sequence that is at least partially complementary to a first nucleotide sequence in the target nucleic acid;
- ii) a second homology arm at or near the 3′ end of the donor DNA, wherein the second homology arm comprises a nucleotide sequence that is at least partially complementary to a second nucleotide sequence in the target nucleic acid;
- and
- iii) a nucleotide sequence of interest between the first homology arm and the second homology arm;
- and
- c) one or more staple oligonucleotides, wherein the one or more staple oligonucleotides are least partially complementary to the nucleotide sequence of interest, such that the one or more staple oligonucleotides hybridize to the nucleotide sequence of interest, such that the single-stranded donor DNA folds into a DNA nanostructure in which the first homology arm and the second homology arm are brought into proximity to one another.
A composition comprising:
- a) a gene-editing polypeptide;
- and
- b) a DNA nanostructure, wherein the DNA nanostructure comprises, in order from 5′ to 3′:
- i) a first homology arm;
- ii) a nucleotide sequence of interest that is hybridized to one or more staple oligonucleotides, wherein the nucleotide sequence of interest is folded via the staple oligonucleotides such that the nucleotide sequence of interest has a length of from about 5 nm to about 500 nm;
- and
- iii) a second homology arm.
A kit comprising:
- a) a gene-editing polypeptide;
- b) a single-stranded donor DNA comprising:
- i) a first homology arm at or near the 5′ end of the donor DNA, wherein the first homology arm comprises a nucleotide sequence that is at least partially complementary to a first nucleotide sequence in the target nucleic acid;
- ii) a second homology arm at or near the 3′ end of the donor DNA, wherein the second homology arm comprises a nucleotide sequence that is at least partially complementary to a second nucleotide sequence in the target nucleic acid;
- and
- iii) a nucleotide sequence of interest between the first homology arm and the second homology arm;
- and
- c) one or more staple oligonucleotides, wherein the one or more staple oligonucleotides are least partially complementary to the nucleotide sequence of interest, such that the one or more staple oligonucleotides hybridize to the nucleotide sequence of interest, such that the donor DNA template folds into a nanostructure in which the first homology arm and the second homology arm are brought into proximity to one another.
Dependencies are inferred from the claim text.