# US 2014/0193915 A1 · Compositions and methods for custom site-specific dna recombinases

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- Number: US 2014/0193915 A1
- Document ID: US20140193915A1
- Status: Published application
- Kind: A1
- Application number: 14/109,823
- Filed: 2013-12-17
- Published: 2014-07-10
- Earliest claimed: 2012-12-18
- Assignees: Monsanto Technology LLC
- Applicants: Monsanto Technology, LLC
- Inventors: Jonathan C. Lamb; Artem G. Evdokimov
- Web page: https://patentgrep.com/patent/US20140193915A1

## CPC

- C12N 9/1241: {Nucleotidyltransferases (2.7.7)}
- C07K 14/4705: {stimulating, promoting or activating activity}
- C12N 15/8213: {Targeted insertion of genes into the plant genome by homologous recombination}
- C12N 15/902: {using homologous recombination}
- C07K 2319/00: Fusion polypeptide
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)

## Abstract

The invention relates to biotechnology and provides novel compositions and methods for sequence-specific or sequence-directed transcription activator-like effector recombinases. The invention also provides novel plant transformation vectors and expression cassettes, which include novel combinations of chimeric recombinases with plant expression and transformation elements. Methods for gene-targeting, DNA sequence removal, and molecular breeding are also provided.

## Claims

### Claim 1

A DNA construct encoding a chimeric recombinase polypeptide comprising a serine recombinase catalytic domain and a TALE DNA-binding domain comprising at least one repeat-variable di-residue (RVD) DNA-binding repeat domain.

### Claim 2 (depends on 1)

The DNA construct of claim 1, comprising a nucleic acid encoding a polypeptide linker, wherein the linker is between the serine recombinase catalytic domain and the TALE DNA-binding domain.

### Claim 3 (depends on 1)

The DNA construct of claim 1, wherein the serine recombinase catalytic domain is positioned at an N-terminal position of the chimeric recombinase polypeptide.

### Claim 4 (depends on 1)

The DNA construct of claim 1, wherein the serine recombinase catalytic domain comprises a small serine recombinase catalytic domain.

### Claim 5 (depends on 4)

The DNA construct of claim 4, wherein the small serine recombinase catalytic domain is a permissive small serine recombinase catalytic domain.

### Claim 6 (depends on 4)

The DNA construct of claim 4, wherein the small serine recombinase catalytic domain is a recombinase selected from the group consisting of Gin20H106Y, GinL7C7-EE2, GinL7C7-EE3, HinC, and HinB (HinH106Y).

### Claim 7 (depends on 1)

The DNA construct of claim 1, wherein the TALE DNA-binding domain is positioned at a C-terminal position of the chimeric recombinase polypeptide.

### Claim 8 (depends on 1)

The DNA construct of claim 1, wherein the TALE DNA-binding domain of said chimeric recombinase polypeptide retains equal or greater binding affinity relative to a TALE DNA-binding domain lacking said recombinase catalytic domain.

### Claim 9 (depends on 1)

The DNA construct of claim 1, wherein a first amino acid of the TALE DNA-binding domain begins 40 to about 160 amino acids upstream from a first amino acid of a first at least one RVD DNA-binding repeat domain.

### Claim 10 (depends on 1)

The DNA construct of claim 1, wherein a first amino acid of the TALE DNA-binding domain begins 53, 81, 85, 98, 102, 113, 117, 130, 136, or 147 amino acids upstream from a first amino acid of a first at least one RVD DNA-binding repeat domain.

### Claim 11 (depends on 2)

The DNA construct of claim 2, wherein the linker comprises at least 2 amino acids.

### Claim 12 (depends on 1)

The DNA construct of claim 1, wherein the chimeric recombinase polypeptide is a permissive recombinase.

### Claim 13 (depends on 2)

The DNA construct of claim 2, wherein the linker comprises about 4 amino acids to about 30 amino acids, wherein the linker is between the serine recombinase catalytic domain and the TALE DNA-binding domain.

### Claim 14 (depends on 12)

The DNA construct of claim 12, wherein the permissive recombinase comprises:
a) a small serine recombinase catalytic domain of the type GinL7C7-EE2 positioned at the N-terminal position; and
b) a TALE DNA-binding domain positioned at the C-terminal position, wherein a first amino acid of the TALE DNA-binding domain begins 136 amino acids upstream from a first amino acid of the at least one RVD DNA-binding repeat domain.

### Claim 15

An isolated nucleic acid sequence encoding a chimeric recombinase DNA-recombination target-sequence comprising, in a 5′ to 3′ orientation:
a) a first TALE binding site;
b) a TALER central sequence comprising, in a 5′ to 3′ orientation:
i) an optional first spacer sequence;
ii) a recombinase core sequence; and
iii) an optional second spacer sequence; and
c) a second TALE binding site.

### Claim 16 (depends on 15)

The isolated nucleic acid sequence of claim 15, wherein the recombinase core sequence comprises at least 8 nucleotides.

### Claim 17 (depends on 15)

The isolated nucleic acid sequence of claim 15, wherein the first and second spacer sequence comprises from about 1 to about 12 nucleotides.

### Claim 18 (depends on 15)

The isolated nucleic acid sequence of claim 15, wherein the first and second spacer sequences comprise a same number of nucleotides.

### Claim 19 (depends on 15)

The isolated nucleic acid sequence of claim 15, wherein the first and second spacer sequences comprise a different number of nucleotides.

### Claim 20 (depends on 1)

A chimeric recombinase polypeptide dimer comprising:
a) a first chimeric recombinase polypeptide according to claim 1 binding with a first chimeric recombinase DNA recombination target sequence; and
b) a second chimeric recombinase polypeptide according to claim 1 binding with the first recombinase DNA recombination target sequence, wherein the first chimeric recombinase polypeptide and the second chimeric recombinase polypeptide form a dimer.

### Claim 21 (depends on 20)

The chimeric recombinase polypeptide dimer of claim 20, wherein each of the chimeric recombinase polypeptides comprises a polypeptide linker.

### Claim 22 (depends on 1)

A chimeric recombinase polypeptide tetramer comprising:
a) a first pair of chimeric recombinase polypeptides according to claim 1 binding with a first chimeric recombinase DNA recombination target sequence; and
b) a second pair of chimeric recombinase polypeptides according to claim 1 binding with a second chimeric recombinase DNA recombination target sequence, wherein the first pair of chimeric recombinase polypeptides and the second pair of chimeric recombinase polypeptides form a tetramer.

### Claim 23 (depends on 22)

The chimeric recombinase polypeptide tetramer of claim 22, wherein each of the chimeric recombinase polypeptides of the first pair of chimeric recombinase polypeptides comprises a polypeptide linker.

### Claim 24 (depends on 22)

The chimeric recombinase polypeptide tetramer of claim 22, wherein each of the chimeric recombinase polypeptides of the second pair of chimeric recombinase polypeptides comprises a polypeptide linker.

### Claim 25 (depends on 1)

A method of gene-targeting comprising binding a chimeric recombinase DNA-recombination target-sequence with at least two chimeric recombinase polypeptides according to claim 1.

### Claim 26 (depends on 25)

The method of claim 25, wherein each of the chimeric recombinase polypeptides comprises a polypeptide linker.

### Claim 27 (depends on 1)

A method of gene-targeting comprising:
a) binding a first pair of chimeric recombinase polypeptides according to claim 1 with a first chimeric recombinase DNA recombination target sequence; and
b) binding a second pair of chimeric recombinase polypeptides with a second chimeric recombinase DNA recombination target sequence, wherein the first pair of chimeric recombinase polypeptides and the second pair of chimeric recombinases polypeptides form a tetramer, and wherein the tetramer mediates recombination between the first and the second chimeric recombinase DNA recombination target sequences.

### Claim 28 (depends on 27)

The method of claim 27, wherein each of the chimeric recombinase polypeptides of the first pair of chimeric recombinase polypeptides comprises a polypeptide linker.

### Claim 29 (depends on 27)

The method of claim 27, wherein each of the chimeric recombinase polypeptides of the second pair of chimeric recombinase polypeptides comprises a polypeptide linker.

### Claim 30 (depends on 1)

The DNA construct of claim 1, wherein the serine recombinase catalytic domain is positioned at a C-terminal position of the chimeric recombinase polypeptide.

### Claim 31 (depends on 30)

The DNA construct of claim 30, comprising a nucleic acid sequence encoding a first polypeptide linker, wherein the first linker is between a C-terminus of the TALE DNA-binding domain and an N-terminus of the serine recombinase catalytic domain.

### Claim 32 (depends on 1)

The DNA construct of claim 1, wherein the TALE DNA-binding domain is positioned at an N-terminal position of the chimeric recombinase polypeptide.

### Claim 33 (depends on 30)

The DNA construct of claim 30, comprising a nucleic acid sequence encoding a non-specific DNA-contact domain polypeptide, wherein the DNA-contact domain polypeptide is positioned at the C-terminus of the serine recombinase catalytic domain.

### Claim 34 (depends on 33)

The DNA construct of claim 33, comprising a nucleic acid sequence encoding a second polypeptide linker, wherein the second linker is between a C-terminus of the serine recombinase catalytic domain and an N-terminus of the non-specific DNA-contact domain.

### Claim 35 (depends on 31)

The DNA construct of claim 31, wherein the first linker comprises at least 2 amino acids.

### Claim 36 (depends on 31)

The DNA construct of claim 31, wherein the first linker comprises SEQ ID NO:77 or SEQ ID NO:78.

### Claim 37 (depends on 34)

The DNA construct of claim 34, wherein the second linker comprises at least 2 amino acids.

### Claim 38 (depends on 34)

The DNA construct of claim 34, wherein the second linker comprises SEQ ID NO:79.

### Claim 39 (depends on 33)

The DNA construct of claim 33, wherein the DNA-contact domain is selected from the group consisting of SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, and SEQ ID NO:83.

### Claim 40 (depends on 30)

The DNA construct of claim 30, wherein a first amino acid of the TALE DNA-binding domain begins 40 to about 160 amino acids upstream from a first amino acid of a first at least one RVD DNA-binding repeat domain.

### Claim 41 (depends on 30)

The DNA construct of claim 30, wherein a first amino acid of the TALE DNA-binding domain begins 53, 81, 85, 98, 102, 113, 117, 130, 136, or 147 amino acids upstream from a first amino acid of a first at least one RVD DNA-binding repeat domain.

### Claim 42 (depends on 30)

The DNA construct of claim 30, wherein a first amino acid of the TALE DNA-binding domain begins 136 amino acids upstream from a first amino acid of a first at least one RVD DNA-binding repeat domain.

### Claim 43 (depends on 30)

The DNA construct of claim 30, wherein the serine recombinase catalytic domain is positioned at the C-terminus of the TALE DNA-binding domain.

### Claim 44 (depends on 30)

The DNA construct of claim 30, wherein the serine recombinase catalytic domain is positioned at a C-terminal truncation site of the TALE DNA-binding domain, wherein the C-terminal truncation site is selected from the group consisting of SEQ ID NO:84, SEQ ID NO:85, and SEQ ID NO:86.

### Claim 45 (depends on 27)

The method of claim 27, wherein the first and second pairs of chimeric recombinase polypeptides comprise N-TALERs.

### Claim 46 (depends on 27)

The method of claim 27, wherein the first and second pairs of chimeric recombinase polypeptides comprise C-TALERs.

### Claim 47 (depends on 27)

The method of claim 27, wherein the first pair of chimeric recombinase polypeptides comprise N-TALERs or C-TALERs, and the second pair of chimeric recombinase polypeptides comprise N-TALERs or C-TALERs.

### Claim 48

A method of gene-targeting in a cell comprising introducing one or more chimeric recombinase polypeptides and one or more gene-targeting donor constructs comprising one or more TALER recombination sites into a cell, wherein a recombinase core sequence of one or more of said TALER recombination sites differs by 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 bp as compared to a recombinase core sequence in a genome of said cell.

### Claim 49 (depends on 48)

The method of claim 48, wherein the recombinase core sequence of one or more TALER recombination sites of the gene-targeting donor construct, or the recombinase core sequence in the genome of said cell, comprises a native or perfect Gin recombination core sequence.

### Claim 50 (depends on 48)

The method of claim 48, wherein the recombinase core sequence of one or more TALER recombination sites of the gene-targeting donor construct, or the recombinase core sequence in the genome of said cell, comprises at least a 12 bp pseudo-palindrome sequence comprising two 5 bp sequences flanking a central di-nucleotide site of recombination.

### Claim 51 (depends on 48)

The method of claim 48, wherein the recombinase core sequence of one or more TALER recombination sites or the recombinase core sequence in the genome of said cell consists of SEQ ID NO:290.

### Claim 52 (depends on 48)

The method of claim 48, wherein the recombinase core sequence of one or more TALER recombination sites or the recombinase core sequence in the genome of said cell is selected from the group consisting of SEQ ID NOs:285, 286, 287, 288, and 289.

### Claim 53 (depends on 48)

The method of claim 48, wherein the recombinase core sequence of one or more recombinase target sites of the gene-targeting donor construct, or the recombinase core sequence in the genome of said cell, comprises at least a 8 bp pseudo-palindrome sequence comprising two 3 bp sequences flanking a central di-nucleotide site of recombination.