# US 2023/0058352 A1 · High Fidelity SpCas9 Nucleases for Genome Modification

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

- Number: US 2023/0058352 A1
- Document ID: US20230058352A1
- Status: Published application
- Kind: A1
- Application number: 17/790,392
- Filed: 2021-03-11
- Published: 2023-02-23
- Earliest claimed: 2020-03-11
- Assignees: Sigma-Aldrich Co.
- Applicants: Sigma-Aldrich Co. LLC
- Inventors: Fuqiang Chen
- Web page: https://patentgrep.com/patent/US20230058352A1

## CPC

- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 15/102: {Mutagenizing nucleic acids}
- C12N 9/226: {Class 2 CAS enzyme complex, e.g. single CAS protein}
- C12N 15/79: Vectors or expression systems specially adapted for eukaryotic hosts
- C07K 2319/00: Fusion polypeptide
- C07K 2319/09: containing a nuclear localisation signal
- 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)

## Abstract

Engineered Cas9 protein variants and systems, nucleic acids encoding said protein variants and systems, and methods of making and using said protein variants and systems for genome modification.

## Claims

### Claim 1

An engineered Streptococcus pyogenes Cas9 (SpCas9) protein variant comprising a mutation at two or more of amino acid positions 526, 562, 652, 661, 691, 780, 810, 848, 855, 1003, and 1060 (with reference to the numbering system of Streptococcus pyogenes Cas9, SpCas9), wherein a Lysine (K) at one or more of the foregoing amino acid positions is changed to a Leucine (L) or Glutamine (Q), and/or an Arginine (R) at one or more of the foregoing amino acid positions is changed to a Leucine (L) or Glutamine (Q).

### Claim 2 (depends on 1)

The SpCas9 protein variant of claim 1, wherein the SpCas9 protein variant comprising a K855L/Q mutation and at least one other mutation at amino acid positions 526, 562, 652, 661, 691, 780, 810, 848, 1003, and 1060 (with reference to the numbering system of Streptococcus pyogenes Cas9, SpCas9).

### Claim 3 (depends on 1)

The engineered SpCas9 protein variant of claim 1, wherein the SpCas9 protein variant comprising a R661L/Q mutation and at least one other mutation at amino acid positions 526, 562, 652, 691, 780, 810, 848, 855, 1003, and 1060 (with reference to the numbering system of Streptococcus pyogenes Cas9, SpCas9).

### Claim 4 (depends on 1)

The engineered SpCas9 protein variant of claim 1, comprising two mutations at two different amino acid positions selected from K526L/Q, K562L/Q, K652L/Q, K810L/Q, K848L/Q, K855L/Q, R661L/Q, R691L/Q, R780L/Q, K1003L/Q, and R1060L/Q (with reference to the numbering system of Streptococcus pyogenes Cas9, SpCas9).

### Claim 5 (depends on 1)

The engineered SpCas9 protein variant of claim 1, comprising three mutations at three different amino acid positions selected from K526L/Q, K562L/Q, K652L/Q, K810L/Q, K848L/Q, K855L/Q, R661L/Q, R691L/Q, R780L/Q, K1003L/Q, and R1060L/Q.

### Claim 6 (depends on 5)

The engineered SpCas9 protein variant of claim 5, wherein the mutations are selected from the following group:
K562L-R661L-K855Q;
K562Q-R661L-K855Q;
K652L-R661L-K855Q;
K652Q-R661L-K855Q;
R661L-K855Q-K1003Q; and
R661L-K855Q-R1060Q.

### Claim 7 (depends on 5)

The engineered SpCas9 protein variant of claim 5, wherein the mutations are selected from the following group:
K562L-R661L-K855Q;
K562Q-R661L-K855Q;
K652L-R661L-K855Q; and
K652Q-R661L-K855Q.

### Claim 8 (depends on 5)

The engineered SpCas9 protein variant of claim 5, wherein the mutations are selected from the following group:
K526L-R661L-K855Q;
R661L-R691L-K855Q;
R661L-R780L-K855Q;
R661L-R780Q-K855Q;
R661L-K810L-K855Q, and
R661L-K848L-K855Q.

### Claim 9 (depends on 5)

The engineered SpCas9 protein variant of claim 5, wherein the mutations are selected from the following group:
K526Q-R661L-K855Q;
R661L-K810Q-K855Q;
R661L-K855Q-K1003L; and
R661L-K855Q-R1060L.

### Claim 10 (depends on 1)

The engineered SpCas9 protein variant of claim 1, further comprising one or more heterologous domains fused to the N terminal end, the C terminal end, an internal location, or combination thereof.

### Claim 11 (depends on 10)

The engineered SpCas9 protein variant of claim 10, wherein the heterologous domain is selected from a nuclear localization signal, a cell-penetrating domain, a marker or reporter domain which facilitates detection, a chromatin modification domain, an epigenetic modification domain, a transcriptional regulation domain, a DNA or RNA deaminase domain, a uracil-DNA-glycosylase domain, a reverse transcriptase domain, a recombinase domain, an RNA aptamer binding domain, and a non-Cas9 nuclease domain.

### Claim 12 (depends on 1)

The engineered SpCas9 protein variant of claim 1, further comprising at least one nuclear localization signal fused to the N terminal end, the C terminal end, an internal location between the N terminal end and the C terminal end, or combination thereof.

### Claim 13 (depends on 1)

The engineered SpCas9 protein variant of claim 1, further comprising at least one mutation in the RuvC domain, and/or at least one mutation in the HNH domain.

### Claim 14 (depends on 13)

The engineered SpCas9 protein variant of claim 13, wherein the at least one mutation in the RuvC domain, if present, comprises at least one mutation selected from D10A, D8A, E762A, and D986A; and wherein the at least one mutation in the HNH domain, if present, comprises at least one mutation selected from H840A, H559A, N854A, N856A, and N863A.

### Claim 15 (depends on 1)

An engineered Cas9 system comprising the engineered SpCas9 protein variant of claim 1 and at least one engineered guide RNA(s), wherein the at least one engineered guide RNA is designed to complex with the engineered SpCas9 protein variant.

### Claim 16 (depends on 1)

A plurality of nucleic acids encoding the engineered SpCas9 protein variant of claim 1.

### Claim 17 (depends on 15)

A plurality of nucleic acids encoding the engineered SpCas9 system of claim 15.

### Claim 18 (depends on 17)

The plurality of nucleic acids of claim 17, the plurality of nucleic acids comprising at least one nucleic acid encoding the engineered SpCas9 protein variant and at least one nucleic acid encoding the engineered guide RNA.

### Claim 19 (depends on 17)

The plurality of nucleic acids of claim 17, wherein at least one nucleic acid is RNA.

### Claim 20 (depends on 17)

The plurality of nucleic acids of claim 17, wherein at least one nucleic acid is DNA.

### Claim 21 (depends on 17)

The plurality of nucleic acids of claim 17, wherein the at least one nucleic acid encoding the engineered SpCas9 protein variant is codon optimized for expression in a eukaryotic cell.

### Claim 22 (depends on 21)

The plurality of nucleic acids of claim 21, wherein the eukaryotic cell is a human cell, a non-human mammalian cell, a non-mammalian vertebrate cell, an invertebrate cell, a plant cell, or a single cell eukaryotic organism.

### Claim 23 (depends on 17)

The plurality of nucleic acids of claim 17, wherein the at least one nucleic acid encoding the engineered guide RNA is DNA.

### Claim 24 (depends on 17)

The plurality of nucleic acids of claim 17, wherein the at least one nucleic acid encoding the engineered SpCas9 protein variant is operably linked to a phage promoter sequence for in vitro RNA synthesis or protein expression in a bacterial cell, and the at least one nucleic acid encoding the engineered guide RNA is operably linked to a phage promoter sequence for in vitro RNA synthesis.

### Claim 25 (depends on 17)

The plurality of nucleic acids of claim 17, wherein the at least one nucleic acid encoding the engineered Cas9 protein variant is operably linked to a eukaryotic promoter sequence for expression in a eukaryotic cell, and the at least one nucleic acid encoding the engineered guide RNA is operably linked to a eukaryotic promoter sequence for expression in a eukaryotic cell.

### Claim 26 (depends on 16)

At least one vector comprising the plurality of nucleic acids of claim 16.

### Claim 27 (depends on 26)

The at least one vector of claim 26, which is a plasmid vector, a viral vector, or a self-replicating viral RNA replicon.

### Claim 28 (depends on 15)

A eukaryotic cell comprising at least one engineered Cas9 system of claim 15.

### Claim 29 (depends on 28)

The eukaryotic cell of claim 28, which is a human cell, a non-human mammalian cell, a plant cell, a non-mammalian vertebrate cell, an invertebrate cell, or a single cell eukaryotic organism.

### Claim 30 (depends on 29)

The eukaryotic cell of claim 29, which is in vivo, ex vivo, or in vitro.

### Claim 31 (depends on 1)

The engineered SpCas9 protein variant of claim 1, which is a Cas9 homologue.

### Claim 32 (depends on 1)

A ribonucleoprotein (RNP) complex comprising the engineered SpCas9 protein variant of claim 1.

### Claim 33 (depends on 1)

A fusion protein comprising the engineered SpCas9 protein variant of claim 1.

### Claim 34 (depends on 1)

A pharmaceutical composition comprising the engineered SpCas9 protein variant of claim 1 and at least one pharmaceutically acceptable excipient.

### Claim 35

A method for modifying the chromosomal sequence of a eukaryotic cell, the method comprising expressing in the eukaryotic cell the engineered SpCas9 protein variant of claim