# US 10,479,982 B2 · Engineered CRISPR-Cas9 nucleases with altered PAM specificity

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- Number: US 10,479,982 B2
- Document ID: US10479982B2
- Status: Granted
- Kind: B2
- Application number: 15/935,334
- Filed: 2018-03-26
- Published: 2018-10-04
- Granted: 2019-11-19
- 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/US10479982B2

## 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 variant Streptococcus pyogenes Cas9 (SpCas9) protein, comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 1, with a mutation at G1218 and optionally at one or more of the following positions: G1104, S1109, L1111, E1219, D1135, N1317, R1335, T1337, wherein the isolated variant SpCas9 protein can interact with a guide RNA and a target DNA.

### Claim 2 (depends on 1)

The isolated variant SpCas9 protein of claim 1, wherein the mutation at G1218 is G1218R.

### Claim 3 (depends on 1)

The isolated variant SpCas9 protein of claim 1, wherein the mutations are:
(i) D1135V/G1218R/R1335Q (VRQ variant);
(ii) D1135N/G1218R/R1335Q (NRQ variant);
(iii) G1218R/D1135Y/R1335Q (YRQ variant);
(iv) D1135V/G1218R/R1335Q/T1337L (VRQL variant);
(v) D1135V/G1218R/R1335Q/T1337M (VRQM variant);
(vi) D1135V/G1218R/R1335Q/T1337E (VRQE variant);
(vii) D1135V/G1218R/R1335Q/T1337Q (VRQQ variant);
(viii) D1135N/G1218R/R1335Q/T1337L (NRQL variant);
(ix) D1135N/G1218R/R1335Q/T1337M (NRQM variant);
(x) D1135N/G1218R/R1335Q/T1337E (NRQE variant);
(xi) D1135N/G1218R/R1335Q/T1337Q (NRQQ variant);
(xii) D1135Y/G1218R/R1335Q/T1337L (YRQL variant);
(xiii) D1135Y/G1218R/R1335Q/T1337M (YRQM variant);
(xiv) D1135V/G1218R/R1335Q/T1337L (YRQQ variant);
(xv) D1135V/G1218R/E1219V/R1335Q/T1337E (VRVQE variant);
(xvi) D1135N/G1218R/E1219V/R1335Q/T1337E (NRVQE variant);
(xvii) D1135Y/G1218R/E1219V/R1335Q/T1337E (YRVQE variant); or
(xviii) D1135N/G1218R/R1335Q/T1337R (NRQR variant).

### Claim 4 (depends on 1)

The isolated variant SpCas9 protein of claim 1, wherein the mutations are:
(i) L1111H/D1135V/G1218R/R1335Q/T1337R (LVRQR variant);
(ii) D1135V/G1218R/N1317K/R1335Q/T1337R (VRKQR variant);
(iii) G1104K/D1135V/G1218R/R1335Q/T1337R (KVRQR variant);
(iv) S1109T/D1135V/G1218R/R1335Q/T1337R (TVRQR variant); or
(v) D1135N/S1136N/G1218R/R1335Q/T1337R (NNRQR variant).

### Claim 5 (depends on 1)

The isolated variant SpCas9 protein of claim 1, further comprising one or more mutations that decrease nuclease activity selected from the group consisting of mutations at D10, E762, D839, H983, and D986; and one more mutations selected from the group consisting of mutations at H840 and N863.

### Claim 6 (depends on 5)

The isolated variant SpCas9 protein of claim 5, wherein the mutations are:
(i) D10A or D10N, and
(ii) H840A, H840N, or H840Y.

### Claim 7 (depends on 1)

The isolated variant SpCas9 protein of claim 1, wherein the isolated variant SpCas9 protein comprises mutations at G1218, D1135, R1335 and T1337.

### Claim 8 (depends on 7)

The isolated variant SpCas9 protein of claim 7, wherein the isolated variant SpCas9 protein comprises mutations selected from the group consisting of: G1218R, D1135Y, D1135N, D1135V, R1335Q, T1337R, T1337E, T1337L, T1337M and T1337Q.

### Claim 9 (depends on 1)

The isolated variant SpCas9 protein of claim 1, comprising an amino acid sequence that has at least 95% sequence identity to the amino acid sequence of SEQ ID NO:1, with a mutation at G1218 and optionally at one or more of the following positions: G1104, 51109, L1111, D1135, 51136, N1317, R1335, and T1337.

### Claim 10 (depends on 1)

A fusion protein comprising the isolated variant SpCas9 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 11 (depends on 10)

The fusion protein of claim 10, wherein the heterologous functional domain is a transcriptional activation domain.

### Claim 12 (depends on 11)

The fusion protein of claim 11, wherein the transcriptional activation domain is from VP64 or NF-κB p65.

### Claim 13 (depends on 10)

The fusion protein of claim 10, wherein the heterologous functional domain is a transcriptional silencer or transcriptional repression domain.

### Claim 14 (depends on 13)

The fusion protein of claim 13, wherein the transcriptional repression domain is a Krueppel-associated box (KRAB) domain, ERF repressor domain (ERD), or mSin3A interaction domain (SID).

### Claim 15 (depends on 13)

The fusion protein of claim 13, wherein the transcriptional silencer is Heterochromatin Protein 1 (HP1).

### Claim 16 (depends on 10)

The fusion protein of claim 10, wherein the heterologous functional domain is an enzyme that modifies the methylation state of DNA.

### Claim 17 (depends on 16)

The fusion protein of claim 16, wherein the enzyme that modifies the methylation state of DNA is a DNA methyltransferase (DNMT) or a ten-eleven translocation (TET) protein.

### Claim 18 (depends on 17)

The fusion protein of claim 17, wherein the TET protein is ten-eleven translocation 1 (TET1).

### Claim 19 (depends on 10)

The fusion protein of claim 10, wherein the heterologous functional domain is an enzyme that modifies a histone subunit.

### Claim 20 (depends on 19)

The fusion protein of claim 19, wherein the enzyme that modifies a histone subunit is a histone acetyltransferase (HAT), histone deacetylase (HDAC), histone methyltransferase (HMT), or histone demethylase.

### Claim 21 (depends on 10)

The fusion protein of claim 10, wherein the heterologous functional domain is a biological tether.

### Claim 22 (depends on 21)

The fusion protein of claim 21, wherein the biological tether is MS2, Csy4 or lambda N protein.

### Claim 23 (depends on 10)

The fusion protein of claim 10, wherein the heterologous functional domain is FokI.

### Claim 24 (depends on 1)

An isolated nucleic acid encoding the isolated variant SpCas9 protein of claim 1.

### Claim 25 (depends on 24)

A vector comprising the isolated nucleic acid of claim 24, optionally operably linked to one or more regulatory domains.

### Claim 26 (depends on 25)

The vector of claim 25, wherein the vector is a prokaryotic vector.

### Claim 27 (depends on 26)

The vector of claim 26, wherein the prokaryotic vector is a plasmid, a shuttle vector, or an insect vector.

### Claim 28 (depends on 25)

The vector of claim 25, wherein the vector is an expression vector.

### Claim 29 (depends on 1)

A host cell expressing the isolated variant SpCas9 protein of claim 1.

### Claim 30

A method of altering the genome of a cell, the method comprising expressing in the cell, or contacting the cell with,
(i) an isolated variant Streptococcus pyogenes Cas9 (SpCas9) protein, comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 1, with a mutation at G1218 and optionally at one or more of the following positions: G1104, S1109, L1111, E1219, D1135, N1317, R1335, T1337, wherein the isolated variant SpCas9 protein can interact with a guide RNA and a target DNA, or
(ii) a fusion protein comprising an isolated variant Streptococcus pyogenes Cas9 (SpCas9) protein fused to a heterologous functional domain, with an optional intervening linker, wherein the linker does not interfere with activity of the fusion protein, wherein the isolated variant SpCas9 protein comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 1, with a mutation at G1218 and optionally at one or more of the following positions: G1104, S1109, L1111, E1219, D1135, N1317, R1335, T1337, and wherein the isolated variant SpCas9 protein can interact with a guide RNA and a target DNA, and
a guide RNA having a region complementary to a selected portion of the genome of the cell.

### Claim 31 (depends on 30)

The method of claim 30, wherein the cell is a stem cell.

### Claim 32 (depends on 30)

The method of claim 30, wherein the cell is an isolated embryonic stem cell, an isolated mesenchymal stem cell, or an induced pluripotent stem cell.

### Claim 33 (depends on 30)

The method of claim 30, wherein the cell is in a living animal or is in an embryo.

### Claim 34

A method of altering a double stranded DNA (dsDNA) molecule, the method comprising contacting the dsDNA molecule with
(i) an isolated variant Streptococcus pyogenes Cas9 (SpCas9) protein, comprising an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 1, with a mutation at G1218 and optionally at one or more of the following positions: G1104, S1109, L1111, E1219, D1135, N1317, R1335, T1337, wherein the isolated variant SpCas9 protein can interact with a guide RNA and a target DNA, or
(ii) a fusion protein comprising an isolated variant Streptococcus pyogenes Cas9 (SpCas9) protein fused to a heterologous functional domain, with an optional intervening linker, wherein the linker does not interfere with activity of the fusion protein, wherein the isolated variant SpCas9 protein comprises an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 1, with a mutation at G1218 and optionally at one or more of the following positions: G1104, S1109, L1111, E1219, D1135, N1317, R1335, T1337, and wherein the isolated variant SpCas9 protein can interact with a guide RNA and a target DNA,
and a guide RNA having a region complementary to a selected portion of the dsDNA molecule.

### Claim 35 (depends on 4)

The method of claim 4, wherein the dsDNA molecule is in vitro.