Engineered transgene integration platform (ETIP) for gene targeting and trait stacking
First Claim
1. A method for producing a transgenic plant cell, the method comprising:
- transforming a plant cell with a donor nucleic acid molecule and a nucleic acid molecule encoding a site-specific nuclease, which is a fusion protein comprising a cleavage domain and a DNA binding domain selected form the group consisting of;
a meganuclease DNA-binding domain, a leucine zipper DNA-binding domain, a transcription activator-like (TAL) DNA-binding domain, a DNA-binding domain from a recombinase, a zinc finger protein DNA-binding domain, and chimeric combinations thereof, wherein the plant cell comprises a polynucleotide integrated at a locus in the genomic DNA of the plant cell, the polynucleotide comprising, in the 5′
to 3′
direction;
a first promoter,a first, non-functional fragment of a first, functional marker gene,at least one recognition site for the site-specific nuclease, anda second, non-functional fragment of a second, functional marker gene, wherein the first promoter is operably-linked to the first, non-functional fragment of the first, functional marker gene, and wherein the first, functional marker gene encodes a different marker from the marker encoded by the second, functional marker gene,wherein the donor nucleic acid molecule comprises a donor polynucleotide comprising, in the 5′
to 3′
direction, a second fragment of the first, functional marker gene, a nucleotide sequence of interest, a second promoter, and a first fragment of the second, functional marker gene, wherein the second promoter is operably-linked to the first fragment of the second, functional marker gene,thereby expressing the site-specific nuclease to introduce a double-strand break into the genomic DNA of the plant cell at the recognition site of the site-specific nuclease, such that repair of the double-strand break results in integration of the donor polynucleotide at the locus to produce the transgenic plant cell,wherein the transgenic plant cell comprises at the locus, in the 5′
to 3′
direction;
the first promoter,the first, functional marker gene,the nucleotide sequence of interest,the second promoter, and the second, functional marker gene, wherein the first promoter is operably-linked to the first, functional marker gene, and wherein the second promoter is operably-linked to the second, functional marker gene.
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Abstract
An Engineered Transgene Integration Platform (ETIP) is described that can be inserted randomly or at targeted locations in plant genomes to facilitate rapid selection and detection of a GOI that is perfectly targeted (both the 5′ and 3′ ends) at the ETIP genomic location. One element in the subject disclosure is the introduction of specific double stranded breaks within the ETIP. In some embodiments, an ETIP is described using zinc finger nuclease binding sites, but may utilize other targeting technologies such as meganucleases, CRISPRs, TALs, or leucine zippers. Also described are compositions of, and methods for producing, transgenic plants wherein the donor or payload DNA expresses one or more products of an exogenous nucleic acid sequence (e.g. protein or RNA) that has been stably-integrated into an ETIP in a plant cell. In embodiments, the ETIP facilitates testing of gene candidates and plant expression vectors from ideation through Development phases.
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Citations
27 Claims
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1. A method for producing a transgenic plant cell, the method comprising:
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transforming a plant cell with a donor nucleic acid molecule and a nucleic acid molecule encoding a site-specific nuclease, which is a fusion protein comprising a cleavage domain and a DNA binding domain selected form the group consisting of;
a meganuclease DNA-binding domain, a leucine zipper DNA-binding domain, a transcription activator-like (TAL) DNA-binding domain, a DNA-binding domain from a recombinase, a zinc finger protein DNA-binding domain, and chimeric combinations thereof, wherein the plant cell comprises a polynucleotide integrated at a locus in the genomic DNA of the plant cell, the polynucleotide comprising, in the 5′
to 3′
direction;a first promoter, a first, non-functional fragment of a first, functional marker gene, at least one recognition site for the site-specific nuclease, and a second, non-functional fragment of a second, functional marker gene, wherein the first promoter is operably-linked to the first, non-functional fragment of the first, functional marker gene, and wherein the first, functional marker gene encodes a different marker from the marker encoded by the second, functional marker gene, wherein the donor nucleic acid molecule comprises a donor polynucleotide comprising, in the 5′
to 3′
direction, a second fragment of the first, functional marker gene, a nucleotide sequence of interest, a second promoter, and a first fragment of the second, functional marker gene, wherein the second promoter is operably-linked to the first fragment of the second, functional marker gene,thereby expressing the site-specific nuclease to introduce a double-strand break into the genomic DNA of the plant cell at the recognition site of the site-specific nuclease, such that repair of the double-strand break results in integration of the donor polynucleotide at the locus to produce the transgenic plant cell, wherein the transgenic plant cell comprises at the locus, in the 5′
to 3′
direction;the first promoter, the first, functional marker gene, the nucleotide sequence of interest, the second promoter, and the second, functional marker gene, wherein the first promoter is operably-linked to the first, functional marker gene, and wherein the second promoter is operably-linked to the second, functional marker gene. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 24, 25, 26, 27)
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20. A method for producing a transgenic plant cell, the method comprising:
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transforming a plant cell with a donor nucleic acid molecule and a nucleic acid molecule encoding a site-specific nuclease, which is a fusion protein comprising a cleavage domain and a DNA binding domain selected form the group consisting of;
a meganuclease DNA-binding domain, a leucine zipper DNA-binding domain, a transcription activator-like (TAL) DNA-binding domain, a DNA-binding domain from a recombinase, a zinc finger protein DNA-binding domain, and chimeric combinations thereof, wherein the plant cell comprises a polynucleotide integrated at a locus in the genomic DNA of the plant cell, the polynucleotide comprising, in the 5′
to 3′
direction;a first incomplete expression cassette consisting of, in the 5′
to 3′
direction;a first promoter, a first, non-functional fragment of a first, functional marker gene operably linked to the first promoter, a first homology arm nucleotide sequence, and a 3′
untranslated region, at least one recognition site for the site-specific nuclease, anda second incomplete expression cassette consisting of, in the 5′
to 3′
direction;a second homology arm nucleotide sequence, a second, non-functional fragment of a second, different functional marker gene, and a 3′
untranslated region,wherein the donor nucleic acid molecule comprises a donor polynucleotide comprising, in the 5′
to 3′
direction;a third incomplete expression cassette consisting of, in the 5′
to 3′
direction;the first homology arm nucleotide sequence, a second fragment of the first, functional marker gene, a 3′
untranslated region,a nucleotide sequence of interest, and a fourth incomplete expression cassette consisting of, in the 5′
to 3′
direction;a second promoter, a first fragment of the second, functional marker gene operably linked to the second promoter, and the second homology arm nucleotide sequence, thereby expressing the site-specific nuclease to introduce a double-strand break into the genomic DNA of the plant cell at the recognition site of the site-specific nuclease, such that repair of the double-strand break results in integration of the donor polynucleotide at the locus to produce the transgenic plant cell, wherein the transgenic plant cell comprises at the locus, in the 5′
to 3′
direction;
the first promoter,the first, functional marker gene containing the first homology arm nucleotide sequence, the nucleotide sequence of interest, the second promoter, and the second, functional marker gene containing the second homology arm, wherein the first promoter is operably-linked to the first, functional marker gene, and wherein the second promoter is operably-linked to the second, functional marker gene. - View Dependent Claims (21, 22, 23)
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Specification