# US 12,006,521 B2 · CRISPR-associated transposases and uses thereof

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- Number: US 12,006,521 B2
- Document ID: US12006521B2
- Status: Granted
- Kind: B2
- Application number: 17/226,264
- Filed: 2021-04-09
- Published: 2021-12-09
- Granted: 2024-06-11
- Earliest claimed: 2015-12-29
- Assignees: Monsanto Technology LLC
- Applicants: Monsanto Technology LLC
- Inventors: Jaishree M. Chittoor; Ervin Nagy
- Web page: https://patentgrep.com/patent/US12006521B2

## CPC

- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 9/222: {Clustered regularly interspaced short palindromic repeats [CRISPR]-associated [CAS] enzymes}
- C12N 15/102: {Mutagenizing nucleic acids}
- C12N 15/11: DNA or RNA fragments; Modified forms thereof (DNA or RNA not used in recombinant technology, C07H21/00); {Non-coding nucleic acids having a biological activity}
- C12N 15/8241: {Phenotypically and genetically modified plants via recombinant DNA technology}
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N 2800/80: Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Provided herein are systems, methods, and compositions for the modification of target DNA sequences. More particularly, systems, methods, and compositions for editing genomic DNA in eukaryotic cells with a CRISPR-associated transposase are provided. Also provided are vectors and vector systems which encode one or more CRISPR-associated transposases, as well as methods for the design and use of such vectors. Also provided are methods for identifying and validating novel CRISPR-associated transposases.

## Claims

### Claim 1

A vector comprising a eukaryotic cell expressible promoter operably linked to a polynucleotide sequence encoding a CRISPR-associated enzyme, wherein the polynucleotide sequence has at least 85% identity to a sequence selected from the group consisting of SEQ ID NOs: 1-10, 12-97, 288-302, 304-594, 606-623, 2020-2069, 2075-2504, 2645-2679, 2700-2749, 2755-3184, and 3325-3359, and wherein the polynucleotide sequence encodes a protein having at least 85% identity to a sequence selected from the group consisting of SEQ ID NOs: 124-133, 135-220, and 277-283.

### Claim 2 (depends on 1)

The vector of claim 1, wherein the vector further comprises at least one polynucleotide sequence encoding a guide RNA operably linked to a second promoter capable of expressing the guide RNA, wherein the guide RNA is capable of forming a complex with the CRISPR-associated enzyme.

### Claim 3 (depends on 1)

The vector of claim 1, wherein the vector is an Agrobacterium vector.

### Claim 4 (depends on 1)

The vector of claim 1, wherein the vector further comprises at least one polynucleotide encoding a donor polynucleotide.

### Claim 5 (depends on 1)

The vector of claim 1, wherein the guide RNA comprises a Puf domain binding site.

### Claim 6 (depends on 2)

The vector of claim 2, wherein the second promoter is a U6 promoter.

### Claim 7 (depends on 1)

The vector of claim 1, wherein the vector further comprises at least one polynucleotide sequence encoding a selectable marker.

### Claim 8 (depends on 1)

A eukaryotic cell comprising the vector of claim 1.

### Claim 9 (depends on 8)

The eukaryotic cell of claim 8, wherein the eukaryotic cell is a plant cell.

### Claim 10

A method for sequence-specific modification of a target nucleic acid sequence in a eukaryotic cell, the method comprising providing to the eukaryotic cell:
(a) a recombinant nucleic acid comprising a heterologous promoter operably linked to a polynucleotide sequence having at least 85% identity to a sequence selected from the group consisting of SEQ ID NOs: 1-10, 12-97, 288-302, 304-594, 606-623, 2020-2069, 2075-2504, 2645-2679, 2700-2749, 2755-3184, and 3325-3359, wherein the polynucleotide sequence encodes a protein having at least 85% identity to a sequence selected from the group consisting of SEQ ID NOs: 124-133, 135-220, and 277-283, and wherein the recombinant nucleic acid expresses an effective amount of a CRISPR-associated enzyme encoded by the polynucleotide sequence; and
(b) a guide RNA capable of hybridizing with the target nucleic acid sequence, wherein the guide RNA forms a complex with the CRISPR-associated enzyme and the complex modifies the target nucleic acid sequence.

### Claim 11 (depends on 10)

The method of claim 10, wherein the guide RNA is provided by expressing in the eukaryotic cell a recombinant DNA molecule encoding the guide RNA.

### Claim 12 (depends on 10)

The method of claim 10, wherein the guide RNA is provided by particle bombardment.

### Claim 13 (depends on 10)

The method of claim 10, wherein the method further comprises providing a donor polynucleotide to the eukaryotic cell.

### Claim 14 (depends on 10)

The method of claim 10, wherein the eukaryotic cell is a plant cell.

### Claim 15 (depends on 14)

The method of claim 14, wherein the plant cell is selected from the group consisting of a corn cell, a soybean cell, an alfalfa cell, a cotton cell, a canola cell, a wheat cell, a rice cell, a broccoli cell, a cauliflower cell, a tomato cell, an eggplant cell, a pepper cell, a lettuce cell, a spinach cell, a strawberry cell, a potato cell, a squash cell, a melon cell, a blueberry cell, a raspberry cell, a blackberry cell, a grape cell, and a carrot cell.

### Claim 16

A recombinant nucleic acid comprising a heterologous promoter operably linked to a polynucleotide sequence encoding a protein with an amino acid sequence selected from the group consisting of SEQ ID NOs: 124-133, 135-220, and 277-283, wherein the polynucleotide sequence is codon optimized for expression in a plant cell.

### Claim 17 (depends on 16)

The recombinant nucleic acid of claim 16, wherein the polynucleotide sequence is selected from the group consisting of SEQ ID NOs: 2020-2069, 2075-2504, 2645-2679, 2700-2749, 2755-3184, and 3325-3359.

### Claim 18 (depends on 16)

The recombinant nucleic acid of claim 16, wherein the plant cell is a corn cell or a soybean cell.

### Claim 19 (depends on 16)

A eukaryotic cell comprising the recombinant nucleic acid of claim 16.

### Claim 20 (depends on 19)

The eukaryotic cell of claim 19, wherein the eukaryotic cell is a plant cell.