Methods to improve site-directed integration frequency
AssigneeMonsanto Technology LLC
InventorsJohn BRADLEY, Larry A. GILBERTSON, Ervin NAGY, Christine SHYU, Peizhen YANG, Xudong YE
Life of this patent
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Abstract
The present disclosure relates to compositions and methods for improving site-directed integration frequency in the genome of plant cells.
Claims128
A recombinant nucleic acid construct comprising:
- (a) a left border sequence;
- (b) a first recombination site and a second recombination site;
- (c) a target site for a guided nuclease;
- (d) a sequence of interest;
- and
- (e) a right border sequence,
- wherein the target site for the guided nuclease and the sequence of interest are positioned between the first recombination site and the second recombination site.
A recombinant nucleic acid construct comprising:
- (a) a left border sequence;
- (b) a first recombination site and a second recombination site;
- (c) a sequence of interest;
- (d) a target site for a guided nuclease;
- (e) a promoter;
- and
- (f) a right border sequence,
- wherein the sequence of interest and the target site for a guided nuclease are positioned between the first recombination site and the second recombination site.
A recombinant nucleic acid construct comprising:
- (a) a left border sequence;
- (b) a first promoter;
- (c) a first recombination site and a second recombination site;
- (d) a target site for the guided nuclease;
- (e) a sequence of interest;
- and
- (f) a right border sequence,
- wherein the target site for a guided nuclease and the sequence of interest are positioned between the first recombination site and the second recombination site.
A recombinant nucleic acid construct comprising:
- (a) a first left border (LB) sequence;
- (b) a first recombination site and a second recombination site;
- (c) a sequence of interest;
- (d) a target site for a guided nuclease;
- (e) a first right border (RB) sequence;
- (f) a second RB sequence;
- and
- (g) a second LB sequence,
- wherein the sequence of interest and the target site for a guided nuclease are positioned between the first recombination site and the second recombination site.
A recombinant nucleic acid construct comprising:
- (a) a left border sequence;
- (b) a sequence encoding a selectable marker;
- (c) a first recombination site and a second recombination site;
- (d) a target site for a guided nuclease;
- (e) a sequence of interest;
- (f) a first promoter;
- and
- (g) a right border sequence,
- wherein the first promoter and the sequence encoding the selectable marker are in the same orientation, and wherein the target site for a guided nuclease and the sequence of interest are positioned between the first recombination site and the second recombination site.
A recombinant nucleic acid construct comprising:
- (a) a left border sequence;
- (b) a sequence encoding at least one guide RNA operably linked to a first promoter;
- (c) a first recombination site and a second recombination site;
- (d) a target site for a guided nuclease;
- (e) a sequence of interest;
- and
- (f) a right border sequence,
- wherein the target site for a guided nuclease and the sequence of interest are positioned between the first recombination site and the second recombination site.
A recombinant nucleic acid construct comprising:
- (a) a left border sequence;
- (b) a first large intergenic region (LIR) and a second LIR;
- (c) a sequence of interest;
- (d) a target site for a guided nuclease;
- and
- (e) a right border sequence,
- wherein the sequence of interest and the target site for a guided nuclease are positioned between the first LIR and the second LIR.
A method comprising introducing to a cell a recombinant DNA construct comprising:
- (a) a left border sequence;
- (b) a first recombination site and a second recombination site;
- (c) a target site for a guided nuclease;
- (d) a sequence of interest;
- and
- (e) a right border sequence,
- wherein the target site for the guided nuclease and the sequence of interest are positioned between the first recombination site and the second recombination site, wherein the guided nuclease cleaves the target site for a guided nuclease and a genomic target site, and wherein the sequence of interest is inserted into the genomic target site.
An engineered double-stranded donor DNA molecule comprising one or more target sites for a guided nuclease adjacent to at least one sequence of interest, wherein the sequence of nucleotide positions 20 to 23 of the target strand within the one or more target sites for a guided nuclease are the reverse complement of the sequence of nucleotide positions 20 to 23 of the target strand within a genomic target site.
The engineered double-stranded donor DNA molecule of claim 118, wherein the sequence of nucleotide positions 1 to 19 of the target strand within the one or more target sites for a guided nuclease are identical to the sequence of nucleotide positions 1 to 19 of the target strand within the genomic target site.
The engineered double-stranded donor DNA molecule of claim 124, wherein the RNA guided nuclease is a Cas12a nuclease.
Dependencies are inferred from the claim text.