# US 12,110,517 B2 · Optimized protein linkers and methods of use

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- Number: US 12,110,517 B2
- Document ID: US12110517B2
- Status: Granted
- Kind: B2
- Application number: 18/334,407
- Filed: 2023-06-14
- Published: 2024-01-04
- Granted: 2024-10-08
- Earliest claimed: 2020-07-21
- Assignees: Pairwise Plants Services, Inc.
- Applicants: Pairwise Plants Services, Inc.
- Inventors: Sharon Leigh Guffy; Joseph Matthew Watts
- Web page: https://patentgrep.com/patent/US12110517B2

## CPC

- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 9/226: {Class 2 CAS enzyme complex, e.g. single CAS protein}
- C07K 14/001: {by chemical synthesis}
- C12N 9/78: acting on carbon to nitrogen bonds other than peptide bonds (3.5)
- 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 15/907: {in mammalian cells}
- C12Y 305/04002: Adenine deaminase (3.5.4.2)
- C12Y 305/04004: Adenosine deaminase (3.5.4.4)
- C07K 2319/00: Fusion polypeptide
- C07K 2319/80: containing a DNA binding domain, e.g. Lacl or Tet-repressor
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- 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)
- C12Y: ENZYMES

## Abstract

The invention relates to peptide linkers and fusion proteins comprising linkers designed for optimizing the activity of the proteins comprised therein, and methods for using the same. The invention further relates to newly designed Cas12a-based adenine base editors.

## Claims

### Claim 1

A Type V Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated (Cas) (CRISPR-Cas) system comprising:
(a) a fusion protein comprising a Cas12a domain, a linker comprising an amino acid sequence of any one of SEQ ID NOs: 1-24, and a polypeptide of interest, wherein the Cas12a domain is linked to the polypeptide of interest via any one of the amino acid sequences of SEQ ID NOs: 1-24, or a nucleic acid encoding the fusion protein; and
(b) a guide nucleic acid comprising a spacer sequence and a repeat sequence, wherein the guide nucleic acid is capable of forming a complex with the Cas12a domain of the fusion protein and the spacer sequence is capable of hybridizing to a target nucleic acid, thereby guiding the Cas12a domain and the polypeptide of interest to the target nucleic acid, whereby the system is capable of modifying or modulating the target nucleic acid.

### Claim 2 (depends on 1)

The system of claim 1, wherein the Cas12a domain comprises a mutation in the nuclease active site.

### Claim 3 (depends on 1)

The system of claim 1, wherein the Cas12a domain is linked at its C-terminus and/or its N-terminus to any one of the amino acid sequences of SEQ ID NOs: 1-24.

### Claim 4 (depends on 1)

The system of claim 1, wherein the Cas12a domain is linked by its C-terminus to the N-terminus of the polypeptide of interest via any one of the amino acid sequences of SEQ ID NOs: 1-24.

### Claim 5 (depends on 1)

The system of claim 1, wherein the Cas12a domain is linked by its N-terminus to the C-terminus of the polypeptide of interest via any one of the amino acid sequences of SEQ ID NOs: 1-24.

### Claim 6 (depends on 1)

The system of claim 1, wherein the polypeptide of interest comprises at least one polypeptide or protein domain having deaminase activity, nickase activity, recombinase activity, transposase activity, methylase activity, glycosylase activity, glycosylase inhibitor activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, nuclease activity, single-strand RNA cleavage activity, double-strand RNA cleavage activity, restriction endonuclease activity, nucleic acid binding activity, methyltransferase activity, DNA repair activity, DNA damage activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer forming activity, integrase activity, transposase activity, polymerase activity, ligase activity, helicase activity, or photolyase activity.

### Claim 7 (depends on 1)

The system of claim 1, wherein the polypeptide of interest is an adenine deaminase or adenine deaminase domain.

### Claim 8 (depends on 7)

The system of claim 7, wherein the adenine deaminase is tRNA-specific adenosine deaminase (TadA).

### Claim 9 (depends on 7)

The system of claim 7, wherein polypeptide of interest is an adenine deaminase dimer.

### Claim 10 (depends on 7)

A method of editing a target nucleic acid, comprising contacting a cell or a cell free system comprising the target nucleic acid with the system of claim 7 under conditions wherein the fusion protein is expressed and forms a complex with the guide nucleic acid, the complex hybridizing to the target nucleic acid, wherein the adenine deaminase domain converts an adenosine (A) to a guanine (G) in the target nucleic acid, thereby editing the target nucleic acid.

### Claim 11 (depends on 9)

The system of claim 9, wherein the adenine deaminase dimer is a first adenine deaminase and a second adenine deaminase.

### Claim 12 (depends on 1)

The system of claim 1, wherein the fusion protein of (a) is encoded by a nucleic acid, and nucleic acid encoding the fusion protein of (a) and/or the guide nucleic acid of (b) are comprised in one or more expression cassettes and/or vectors.

### Claim 13 (depends on 1)

The system of claim 1, wherein the polypeptide of interest is a
(a) a first adenine deaminase domain, and
(b) a second adenine deaminase domain,
wherein the Cas12a domain is linked to the first adenine deaminase domain or the second adenine deaminases domain via any one of the amino acid sequence of SEQ ID NOs: 1-24.

### Claim 14 (depends on 13)

A cell comprising the system of claim 13, wherein the cell is from an animal, a plant, a fungus, an archaeon, or a bacterium.

### Claim 15 (depends on 13)

A method of editing a target nucleic acid, comprising contacting a cell or a cell free system comprising the target nucleic acid with the system of claim 13 under conditions wherein the fusion protein is expressed and forms a complex with the guide nucleic acid, the complex hybridizing to the target nucleic acid, wherein the adenine deaminase domain converts an adenosine (A) to a guanine (G) in the target nucleic acid, thereby editing the target nucleic acid.

### Claim 16 (depends on 1)

The system of claim 1, wherein the polypeptide of interest is a
(a) a first adenine deaminase domain, and
(b) a second adenine deaminase domain, wherein the first adenine deaminase is a wild type adenine deaminase and the second adenine deaminase domain is a mutated/evolved adenine deaminase domain and the C-terminus of the Cas12a domain is linked to the N-terminus of the second adenine deaminase domain via any one of the amino acid sequences of SEQ ID NOs: 11-15; the C-terminus of the Cas12a domain is linked to the N-terminus of the first adenine deaminase domain via any one of the amino acid sequences of SEQ ID NOs: 11-24; or the N-terminus of the Cas12a domain is linked to the C-terminus of the second adenine deaminase domain via any one of the amino acid sequences of SEQ ID NOs: 1-10.

### Claim 17 (depends on 16)

A cell comprising the system of claim 16, wherein the cell is from an animal, a plant, a fungus, an archaeon, or a bacterium.

### Claim 18 (depends on 16)

A method of editing a target nucleic acid, comprising contacting a cell or a cell free system comprising the target nucleic acid with the system of claim 16 under conditions wherein the fusion protein is expressed and forms a complex with the guide nucleic acid, the complex hybridizing to the target nucleic acid, wherein the adenine deaminase domain converts an adenosine (A) to a guanine (G) in the target nucleic acid, thereby editing the target nucleic acid.

### Claim 19 (depends on 1)

The system of claim 1, wherein the Cas12a domain comprises a mutation in the nuclease active site and the polypeptide of interest is a
(a) a first adenine deaminase domain; and
(b) a second adenine deaminase domain;
wherein the second adenine deaminase domain is different from the first adenine deaminase domain, the C-terminus of the first adenine deaminase domain is linked to the N-terminus of the second deaminase domain, and the N-terminus of the Cas12a domain is linked to the C-terminus of the second adenine deaminase domain via any one of the amino acid sequences of SEQ ID NOs: 1-10.

### Claim 20 (depends on 19)

A cell comprising the system of claim 19, wherein the cell is from an animal, a plant, a fungus, an archaeon, or a bacterium.

### Claim 21 (depends on 19)

A method of editing a target nucleic acid, comprising contacting a cell or a cell free system comprising the target nucleic acid with the system of claim 19 under conditions wherein the fusion protein is expressed and forms a complex with the guide nucleic acid, the complex hybridizing to the target nucleic acid, wherein the adenine deaminase domain converts an adenosine (A) to a guanine (G) in the target nucleic acid, thereby editing the target nucleic acid.

### Claim 22 (depends on 1)

A cell comprising the system of claim 1, wherein the cell is from an animal, a plant, a fungus, an archaeon, or a bacterium.