# US 12,180,520 B2 · Engineered CRISPR-Cas9 nucleases with altered PAM specificity

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- Number: US 12,180,520 B2
- Document ID: US12180520B2
- Status: Granted
- Kind: B2
- Application number: 17/010,107
- Filed: 2020-09-02
- Published: 2020-12-24
- Granted: 2024-12-31
- Earliest claimed: 2015-03-03
- Assignees: The General Hospital Corporation
- Applicants: The General Hospital Corporation
- Inventors: J. Keith Joung; Benjamin Kleinstiver
- Web page: https://patentgrep.com/patent/US12180520B2

## CPC

- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 9/226: {Class 2 CAS enzyme complex, e.g. single CAS protein}
- C12N 15/102: {Mutagenizing nucleic acids}
- C12N 15/111: {General methods applicable to biologically active non-coding nucleic acids}
- C12N 15/63: Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N 15/90: Stable introduction of foreign DNA into chromosome
- C12Y 301/00: Hydrolases acting on ester bonds (3.1)
- A01K 2227/40: Fish
- 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)
- C07K 2319/71: containing domain for transcriptional activaation, e.g. VP16
- 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 2800/22: Vectors comprising a coding region that has been codon optimised for expression in a respective host
- 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)
- C12Y: ENZYMES
- A01K: ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- 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

Engineered CRISPR-Cas9 nucleases with altered and improved PAM specificities and their use in genomic engineering, epigenomic engineering, and genome targeting.

## Claims

### Claim 1

A complex comprising:
a catalytically inactive Staphylococcus aureus Cas9 (SaCas9) protein comprising an amino acid sequence that has at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 2, wherein the amino acid corresponding to E782 of SEQ ID NO: 2, the amino acid corresponding to N968 of SEQ ID NO: 2, and/or the amino acid corresponding to R1015 of SEQ ID NO: 2 is mutated in the SaCas9 protein, and
a guide RNA having a region complementary to a selected portion of a genome of the cell, wherein the SaCas9 protein is capable of binding to the guide RNA.

### Claim 2 (depends on 1)

The complex of claim 1, wherein the amino acid corresponding to E782 is mutated to K, the amino acid corresponding to N968 is mutated to K, and/or the amino acid corresponding to R1015 is mutated to Q or H in the SaCas9 protein.

### Claim 3 (depends on 1)

The complex of claim 1, wherein the amino acid corresponding to E782 is mutated to K, the amino acid corresponding to N968 is mutated to K, and the amino acid corresponding to R1015 is mutated to Q or H in the SaCas9 protein, and optionally wherein the amino acid corresponding to E735 of SEQ ID NO: 2 is mutated to K, the amino acid corresponding to K929 of SEQ ID NO: 2 is mutated to R, the amino acid corresponding to A1021 of SEQ ID NO: 2 is mutated to T, or the amino acid corresponding to K1044 of SEQ ID NO: 2 is mutated to N in the SaCas9 protein.

### Claim 4 (depends on 1)

The complex of claim 1, wherein the amino acid corresponding to D10 of SEQ ID NO: 2, the amino acid corresponding to D556 of SEQ ID NO: 2, the amino acid corresponding to H557 of SEQ ID NO: 2, and/or the amino acid corresponding to N580 of SEQ ID NO: 2 is mutated in the SaCas9 protein.

### Claim 5 (depends on 4)

The complex of claim 4, wherein the amino acid corresponding to D10 is mutated to A, the amino acid corresponding to D556 is mutated to A, the amino acid corresponding to H557 is mutated to A, and/or the amino acid corresponding to N580 is mutated to A in the SaCas9 protein.

### Claim 6 (depends on 4)

The complex of claim 4, wherein
(i) the amino acid corresponding to D10 is mutated to A and the amino acid corresponding to H557 is mutated to A, or
(ii) the amino acid corresponding to D10 is mutated to A, the amino acid corresponding to D556 is mutated to A, the amino acid corresponding to H557 is mutated to A, and the amino acid corresponding to N580 is mutated to A in the SaCas9 protein.

### Claim 7 (depends on 1)

The complex of claim 1, wherein the SaCas9 protein is fused to a heterologous functional domain.

### Claim 8 (depends on 7)

The complex of claim 7, wherein the heterologous functional domain is a transcriptional activation domain.

### Claim 9 (depends on 8)

The complex of claim 8, wherein the transcriptional activation domain is from VP64 or NF-κB p65.

### Claim 10 (depends on 7)

The complex of claim 7, wherein the heterologous functional domain is a transcriptional silencer or transcriptional repression domain.

### Claim 11 (depends on 10)

The complex of claim 10, wherein the transcriptional repression domain is a Krueppel-associated box (KRAB) domain, ERF repressor domain (ERD), or mSin3A interaction domain (SID).

### Claim 12 (depends on 10)

The complex of claim 10, wherein the transcriptional silencer is Heterochromatin Protein 1 (HP1).

### Claim 13 (depends on 7)

The complex of claim 7, wherein the heterologous functional domain is an enzyme that modifies the methylation state of DNA.

### Claim 14 (depends on 13)

The complex of claim 13, wherein the enzyme that modifies the methylation state of DNA is a DNA methyltransferase (DNMT) or a Ten-Eleven-Translocation (TET) protein.

### Claim 15 (depends on 14)

The complex of claim 14, wherein the TET protein is Ten-Eleven-Translocation 1 protein (TET1).

### Claim 16 (depends on 7)

The complex of claim 7, wherein the heterologous functional domain is an enzyme that modifies a histone subunit.

### Claim 17 (depends on 16)

The complex of claim 16, wherein the enzyme that modifies a histone subunit is a histone acetyltransferase (HAT), a histone deacetylase (HDAC), a histone methyltransferase (HMT), or a histone demethylase.

### Claim 18 (depends on 7)

The complex of claim 7, wherein the heterologous functional domain is a biological tether.

### Claim 19 (depends on 18)

The complex of claim 18, wherein the biological tether is MS2, Csy4, or lambda N protein.

### Claim 20 (depends on 7)

The complex of claim 7, wherein the heterologous functional domain is FokI.