# US 2020/0399620 A1 · Engineered CRISPR-Cas9 nucleases with Altered PAM Specificity

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- Number: US 2020/0399620 A1
- Document ID: US20200399620A1
- Status: Published application
- Kind: A1
- Application number: 17/010,107
- Filed: 2020-09-02
- Published: 2020-12-24
- 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/US20200399620A1

## 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

An isolated Staphylococcus aureus Cas9 (SaCas9) protein with mutations at one or more of the following positions: E735, E782, K929, N968, A1021, K1044 and/or R1015.

### Claim 2 (depends on 1)

The isolated protein of claim 1, comprising a sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:2.

### Claim 3 (depends on 1)

The isolated protein of claim 1, comprising one or more of the following mutations:
R1015Q, R1015H, E782K, N968K, E735K, K929R, A1021T, K1044N,
E782K/N968K/R1015H (KKH variant); E782K/K929R/R1015H (KRH variant); or
E782K/K929R/N968K/R1015H (KRKH variant).

### Claim 4 (depends on 1)

The isolated protein of claim 1, further comprising one or more mutations that decrease nuclease activity selected from the group consisting of mutations at one or more of D10, D556, H557, and/or N580.

### Claim 5 (depends on 4)

The isolated protein of claim 4, wherein the mutations are one or more of D10A, D556A, H557A, or N580A.

### Claim 6 (depends on 4)

The isolated protein of claim 4, wherein the mutations are:
(i) D10A/H557A, or
(ii) D10A/D556A/H557A/N580A.

### Claim 7 (depends on 1)

A fusion protein comprising the isolated protein of claim 1, fused to a heterologous functional domain, with an optional intervening linker, wherein the linker does not interfere with activity of the fusion protein.

### Claim 8 (depends on 7)

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

### Claim 9 (depends on 8)

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

### Claim 10 (depends on 7)

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

### Claim 11 (depends on 10)

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

### Claim 13 (depends on 7)

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

### Claim 14 (depends on 13)

The fusion protein of claim 13, wherein the enzyme that modifies the methylation state of DNA is a DNA methyltransferase (DNMT) or a TET protein.

### Claim 15 (depends on 14)

The fusion protein of claim 14, wherein the TET protein is TET1.

### Claim 16 (depends on 7)

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

### Claim 17 (depends on 7)

The fusion protein of claim 7, wherein the enzyme that modifies a histone subunit is a histone acetyltransferase (HAT), histone deacetylase (HDAC), histone methyltransferase (HMT), or histone demethylase.

### Claim 18 (depends on 7)

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

### Claim 19 (depends on 18)

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

### Claim 20 (depends on 7)

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

### Claim 21 (depends on 1)

An isolated nucleic acid encoding the protein of claim 1.

### Claim 22 (depends on 21)

A vector comprising the isolated nucleic acid of claim 21.

### Claim 23 (depends on 21, 22)

The vector of claim 22, wherein the isolated nucleic acid of claim 21 is operably linked to one or more regulatory domains for expressing an isolated Staphylococcus aureus Cas9 (SaCas9) protein with mutations at one or more of the following positions: E735, E782, K929, N968, A1021, K1044 and/or R1015.

### Claim 24 (depends on 23)

A host cell comprising the nucleic acid of claim 23.

### Claim 25 (depends on 1)

A method of altering the genome of a cell, the method comprising expressing in the cell, or contacting the cell with, the isolated protein or fusion protein of claim 1, and a guide RNA having a region complementary to a selected portion of the genome of the cell.

### Claim 26 (depends on 25)

The method of claim 25, wherein the isolated protein or fusion protein comprises one or more of a nuclear localization sequence, cell penetrating peptide sequence, and/or affinity tag.

### Claim 27 (depends on 25)

The method of claim 25, wherein the cell is a stem cell.

### Claim 28 (depends on 27)

The method of claim 27, wherein the cell is an embryonic stem cell, mesenchymal stem cell, or induced pluripotent stem cell; is in a living animal; or is in an embryo.

### Claim 29 (depends on 1)

A method of altering a double stranded DNA (dsDNA) molecule, the method comprising contacting the dsDNA molecule with the isolated protein or fusion protein of claim 1, and a guide RNA having a region complementary to a selected portion of the dsDNA molecule.

### Claim 30 (depends on 29)

The method of claim 29, wherein the dsDNA molecule is in vitro.