# US 2023/0056852 A1 · Cas9 variants and uses thereof

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 2023/0056852 A1
- Document ID: US20230056852A1
- Status: Published application
- Kind: A1
- Application number: 17/937,203
- Filed: 2022-09-30
- Published: 2023-02-23
- Earliest claimed: 2013-09-06
- Assignees: President and Fellows of Harvard College
- Applicants: President and Fellows of Harvard College
- Inventors: David R. Liu; John Paul Guilinger; David B. Thompson
- Web page: https://patentgrep.com/patent/US20230056852A1

## CPC

- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 9/222: {Clustered regularly interspaced short palindromic repeats [CRISPR]-associated [CAS] enzymes}
- A61K 38/465: {acting on ester bonds (3.1), e.g. lipases, ribonucleases}
- C07K 14/315: from Streptococcus (G), e.g. Enterococci
- C12N 9/1241: {Nucleotidyltransferases (2.7.7)}
- C12N 15/01: Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
- C12N 15/907: {in mammalian cells}
- A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00)
- A61K 47/00: Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- C07K 2319/80: containing a DNA binding domain, e.g. Lacl or Tet-repressor
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- 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

Some aspects of this disclosure provide compositions, methods, and kits for improving the specificity of RNA-programmable endonucleases, such as Cas9. Also provided are variants of Cas9, e.g., Cas9 dimers and fusion proteins, engineered to have improved specificity for cleaving nucleic acid targets. Also provided are compositions, methods, and kits for site-specific nucleic acid modification using Cas9 fusion proteins (e.g., nuclease-inactivated Cas9 fused to a nuclease catalytic domain or a recombinase catalytic domain). Such Cas9 variants are useful in clinical and research settings involving site-specific modification of DNA, for example, genomic modifications.

## Claims

### Claim 1

A polypeptide comprising a sequence selected from the group consisting of SGSETPGTSESATPES (SEQ ID NO:16), SGSETPGTSESA (SEQ ID NO:17), and SGSETPGTSESATPEGGSGGS (SEQ ID NO:18).

### Claim 2 (depends on 1)

The polypeptide of claim 1, wherein the sequence is SGSETPGTSESATPES (SEQ ID NO:16).

### Claim 3 (depends on 1)

The polypeptide of claim 1, wherein the sequence is SGSETPGTSESA (SEQ ID NO:17).

### Claim 4 (depends on 1)

The polypeptide of claim 1, wherein the sequence is SGSETPGTSESATPEGGSGGS (SEQ ID NO:18).

### Claim 5 (depends on 1)

The polypeptide of claim 1, wherein the sequence is fused to a peptide domain.

### Claim 6 (depends on 1)

The polypeptide of claim 1, wherein the sequence separates a first peptide domain from a second peptide domain.

### Claim 7 (depends on 6)

The polypeptide of claim 6, wherein the first peptide domain comprises an RNA-programmable domain.

### Claim 8 (depends on 7)

The polypeptide of claim 7, wherein the RNA-programmable domain comprises a Cas9 domain, a nuclease-inactivated Cas9 (dCas9) domain, or a Cas9 nickase domain.

### Claim 9 (depends on 1)

The polypeptide of claim 1, wherein the polypeptide further comprises a nuclear localization signal (NLS) domain.

### Claim 10 (depends on 7)

The polypeptide of claim 7, wherein the polypeptide further comprises a nuclear localization signal (NLS) domain.

### Claim 11 (depends on 7)

The polypeptide of claim 7, wherein the RNA-programmable domain comprises an amino acid sequence that is at least about 80% identical to SEQ ID NO: 2, 4, or 5.

### Claim 12 (depends on 11)

The polypeptide of claim 11, wherein the RNA-programmable domain further comprises amino acid substitutions at positions corresponding to D10 and H840 of SEQ ID NO: 5.

### Claim 13 (depends on 12)

The polypeptide of claim 12, wherein the amino acid substitutions at positions corresponding to D10 and H840 of SEQ ID NO: 5 are either D10A or H840A.

### Claim 14 (depends on 12)

The polypeptide of claim 12, wherein the amino acid substitutions at positions corresponding to D10 and H840 of SEQ ID NO: 5 are D10A and H840A.

### Claim 15 (depends on 7)

The polypeptide of claim 7, wherein the RNA-programmable domain binds a gRNA.

### Claim 16 (depends on 7)

The polypeptide of claim 7, wherein the polypeptide comprises the structure [the RNA-programmable domain]-[the second peptide domain] or [the second peptide domain]-[the RNA-programmable domain], wherein the “-” represents the sequence.

### Claim 17 (depends on 7)

A complex comprising the polypeptide of claim 7 and a gRNA.

### Claim 18 (depends on 7)

A polynucleotide encoding the polypeptide of claim 7.

### Claim 19 (depends on 18)

A vector comprising a polynucleotide of claim 18.

### Claim 20 (depends on 7)

A cell comprising a genetic construct for expressing the polypeptide of claim 7.

### Claim 21 (depends on 20)

The cell of claim 20, wherein the cell is a prokaryotic cell.

### Claim 22 (depends on 20)

The cell of claim 20, wherein the cell is a eukaryotic cell.

### Claim 23 (depends on 22)

The cell of claim 22, wherein the cell is in a human.

### Claim 24 (depends on 7)

A pharmaceutical composition comprising the polypeptide of claim 7 and a pharmaceutically acceptable excipient.

### Claim 25 (depends on 7)

A kit comprising the polypeptide of claim 7, a polynucleotide encoding the polypeptide of claim 7, a vector comprising a polynucleotide encoding the polypeptide of claim 7, a cell comprising a genetic construct for expressing the polypeptide of claim 7, a complex comprising the polypeptide of claim 7 and a gRNA, and/or one or more gRNAs and/or vectors for expressing one or more gRNAs.