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US 12,590,300 B2Granted
Transcription activator-like effectors fused to inteins
AssigneeCibus US LLC
InventorsRobert W. Williams, Sara Wixon
Life of this patent
Estimating the term…
Abstract
Embodiments of the present disclosure are directed to a plurality of nucleotide sequences encoding a first intein fused to at least a portion of a first transcription activator-like effector (TALE), a second nucleotide sequence encoding the first intein fused to at least a portion of a second TALE, and a third nucleotide sequence encoding a second intein fused to at least a portion of a rare-cutting nuclease.
Claims20
A plurality of nucleotide sequences, comprising:
- a first nucleotide sequence encoding a first of a first intein fused to at least a portion of a first transcription activator-like effector (TALE);
- a second nucleotide sequence encoding a second of the first intein fused to at least a portion of a second TALE, wherein the first TALE includes a first plurality of TALE repeat sequences that, in combination, are configured to bind to a first nucleotide sequence in a target DNA sequence, and the second TALE includes a second plurality of TALE repeat sequences that, in combination, are configured to bind to a second nucleotide sequence in the target DNA sequence, and wherein:
- the first nucleotide sequence encodes the first of the first intein comprising an N-terminal intein fused to a C-terminal of the first TALE, and the second nucleotide sequence encodes the second of the first intein comprising the N-terminal intein fused to a C-terminal of the second TALE, or
- the first nucleotide sequence encodes the first of the first intein comprising a C-terminal intein fused to an N-terminal of the first TALE, and the second nucleotide sequence encodes the second of the first intein comprising the C-terminal intein fused to an N-terminal of the second TALE;
- and
- a third nucleotide sequence encoding a second intein fused to at least a portion of a rare-cutting nuclease, wherein the third nucleotide sequence encodes the second intein comprising:
- a C-terminal intein fused to an N-terminal of the at least portion of the rare-cutting nuclease or between portions of the rare-cutting nuclease, or
- an N-terminal intein fused to a C-terminal of the at least portion of the rare-cutting nuclease or between portions of the rare-cutting nuclease, and
- wherein:
- the rare-cutting nuclease includes FokI,
- each of the first nucleotide sequence, the second nucleotide sequence, and the third nucleotide sequence respectively further encode a promoter and a terminator, and
- the first inteins and the second intein include trans-splicing inteins.
9 more elements
A method of transforming a cell, the method comprising:
- contacting a cell with:
- a first nucleotide sequence encoding a first of a first intein fused to at least a portion of a first transcription activator-like effector (TALE) or a product produced from the first nucleotide sequence;
- a second nucleotide sequence encoding a second of the first intein fused to at least a portion of a second TALE or a product produced from the second nucleotide sequence, wherein the first TALE includes a first plurality of TALE repeat sequences that, in combination, bind to a first nucleotide sequence in a target DNA sequence, and the second TALE includes a second plurality of TALE repeat sequences that, in combination, bind to a second nucleotide sequence in the target DNA sequence, and wherein:
- the first nucleotide sequence encodes the first of the first intein comprising an N-terminal intein fused to a C-terminal of the first TALE, and the second nucleotide sequence encodes the second of the first intein comprising the N-terminal intein fused to a C-terminal of the second TALE, or
- the first nucleotide sequence encodes the first of the first intein comprising a C-terminal intein fused to an N-terminal of the first TALE, and the second nucleotide sequence encodes the second of the first intein comprising the C-terminal intein fused to an N-terminal of the second TALE;
- and
- a third nucleotide sequence encoding a second intein fused to at least a portion of a rare-cutting nuclease or a product produced from the third nucleotide sequence wherein the first inteins and the second intein include trans-splicing inteins and the third nucleotide sequence encodes the second intein comprising:
- a C terminal intein fused to an N-terminal of the at least portion of the care-cutting nuclease or between portions of the rare-cutting nuclease;
- or
- an N-terminal intein fused to a C-terminal of the at least portion of the rare-cutting nuclease or between portions of the rare-cutting nuclease, and
- wherein the rare-cutting nuclease includes FokI and each of the first nucleotide sequence, the second nucleotide sequence, and the third nucleotide sequence respectively further encode a promoter and a terminator;
- and
- in response to contacting the cell, splicing the first TALE, the second TALE, and the rare-cutting nuclease by the first of the first intein and the second of the first intein to ones of the second intein to form:
- a first half transcription activator-like effector nuclease (TALEN) including the first TALE and the rare-cutting nuclease;
- and
- a second half TALEN including the second TALE and the rare-cutting nuclease.
13 more elements
An expression construct, comprising:
- a first nucleotide sequence encoding a first of a first intein fused to at least a first transcription activator-like effector (TALE);
- a second nucleotide sequence encoding a second of the first intein fused to at least a second TALE, wherein the first and the second of the first inteins include trans-splicing inteins, wherein the first TALE includes a first plurality of TALE repeat sequences that, in combination, are configured to bind to a first nucleotide sequence in a target DNA sequence, and the second TALE includes a second plurality of TALE repeat sequences that, in combination, are configured to bind to a second nucleotide sequence in the target DNA sequence and wherein:
- the first nucleotide sequence encodes the first of the first intein comprising an N-terminal intein fused to a C-terminal of the first TALE, and the second nucleotide sequence encodes the second of the first intein comprising the N-terminal intein fused to a C-terminal of the second TALE, or
- the first nucleotide sequence encodes the first of the first intein comprising a C-terminal intein fused to an N-terminal of the first TALE, and the second nucleotide sequence encodes the second of the first intein comprising the C-terminal intein fused to an N-terminal of the second TALE;
- and
- a third nucleotide sequence encoding a second intein fused to at least a portion of a rare-cutting nuclease, wherein the second intein comprises a trans-splicing intein and the third nucleotide sequence encodes the second intein comprising:
- a C-terminal intein fused to an N-terminal of the at least portion of the rare-cutting nuclease or between portions of the rare-cutting nuclease;
- or
- an N-terminal intein fused to a C-terminal of the at least portion of the rare-cutting nuclease or between portions of the rare-cutting nuclease, and
- wherein the rare-cutting nuclease includes FokI, and each of the first nucleotide sequence, the second nucleotide sequence, and the third nucleotide sequence further encode a promoter and a terminator.
7 more elements
Dependencies are inferred from the claim text.