Methods and compositions for nucleic acid integration
AssigneeThe Regents of the University of California
InventorsJames K. Nunez, Jennifer A. Doudna
Life of this patent
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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.
Claims16
A method of nucleic acid integration, the method comprising:
- contacting in vitro a target supercoiled DNA molecule with:
- (1) a double stranded donor DNA molecule and a Cas1 protein, or (2) the double stranded donor DNA molecule, the Cas1 protein, and a Cas2 protein;
- wherein:
- (a) the target supercoiled DNA molecule comprises an AT-rich region upstream of a region that forms a DNA cruciform structure;
- and
- (b) the double stranded donor DNA molecule is linear and comprises a 3′-OH overhang with a length of from 1 to 5 nucleotides, wherein at least one 3′ most nucleotide of the double stranded donor DNA molecule is a C,
- wherein said contacting is not in a bacterial or archaeal cell, and wherein the Cas1 protein, or the Cas1 protein complexed with the Cas2 protein, catalyzes preferentially full integration of the C 3′-OH double stranded donor DNA molecule into the target strand of DNA molecule that runs 5′-to-3′.
Dependencies are inferred from the claim text.