Methods and compositions for nucleic acid integration
AssigneeThe Regents of the University of California
InventorsJames K. Nunez, Jennifer A. Doudna
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Abstract
The disclosure provides methods and compositions for the integration (insertion) of a donor DNA molecule into a target DNA molecule. In general, the methods include contacting a target DNA molecule with a linear donor DNA molecule and a Cas 1 protein, where the target DNA molecule includes an AT-rich region (e.g., in some cases positioned 5 and within 50 nucleotides of a region that forms a DNA cruciform structure), where the contacting is not in a bacterial or archaeal cell (e.g., the contacting is in vitro outside of a cell, inside of a eukaryotic cell, etc.), and provides for integration of the donor DNA molecule into the target DNA molecule.
Claims61
A method of nucleic acid integration, the method comprising:
- contacting a target DNA molecule with a donor DNA molecule and a Cas1 protein, wherein:
- (a) the target DNA molecule comprises an AT-rich region;
- and
- (b) the donor DNA molecule is linear,
- wherein said contacting is not in a bacterial or archaeal cell, and provides for integration of the donor DNA molecule into the target DNA molecule.
A composition comprising:
- (a) a Cas1 protein or a nucleic acid comprising a nucleotide sequence that encodes a Cas1 protein;
- and
- (b) at least one of:
- (i) a donor DNA molecule, wherein the donor DNA molecule is a linear DNA molecule;
- and
- (ii) a Cas2 protein or a nucleic acid comprising a nucleotide sequence that encodes a Cas2 protein.
A kit comprising:
- (a) a Cas1 protein or a nucleic acid comprising a nucleotide sequence that encodes a Cas1 protein;
- and
- (b) at least one of:
- (i) a donor DNA molecule, wherein the donor DNA molecule is a linear DNA molecule;
- and
- (ii) a Cas2 protein or a nucleic acid comprising a nucleotide sequence that encodes a Cas2 protein.
- wherein (a) and (b) are present in the same or separate containers.
An isolated Cas1 protein, or a nucleic acid encoding a Cas1 protein, wherein the Cas1 protein comprises an amino acid sequence modification relative to the corresponding naturally occurring Cas1 protein.
An isolated Cas2 protein, or a nucleic acid encoding a Cas2 protein, wherein the Cas2 protein comprises an amino acid sequence modification relative to the corresponding naturally occurring Cas2 protein.
Dependencies are inferred from the claim text.