# US 2025/0154479 A1 · Streptococcus pyogenes-derived cas9 variant

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- Number: US 2025/0154479 A1
- Document ID: US20250154479A1
- Status: Published application
- Kind: A1
- Application number: 18/832,216
- Filed: 2023-01-20
- Published: 2025-05-15
- Earliest claimed: 2022-01-24
- Assignees: Toolgen Incorporated
- Applicants: Toolgen Incorporated
- Inventors: Younghoon KIM; Keunwoo CHOI; Jeongjoon LEE
- Web page: https://patentgrep.com/patent/US20250154479A1

## CPC

- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 9/226: {Class 2 CAS enzyme complex, e.g. single CAS protein}
- C12N 15/10: Processes for the isolation, preparation or purification of DNA or RNA (chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34)
- C12N 15/111: {General methods applicable to biologically active non-coding nucleic acids}
- C12N 15/113: Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)}
- C12N 15/90: Stable introduction of foreign DNA into chromosome
- 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

The present disclosure discloses a SpCas9 variant. The SpCas9 variant is characterized by the ability to recognize a PAM sequence different from that of a wild-type SpCas9 protein.

## Claims

### Claim at position 1

-18. (canceled)

### Claim 19

A Streptococcus pyogenes Cas9 (SpCas9) variant comprising an amino acid sequence with six or more amino acid residue differences compared to SEQ ID NO: 1 which is an amino acid sequence of wild-type SpCas9 protein,
wherein the SpCas9 variant comprises one selected from the following mutations, compared to the wild-type SpCas9 protein:
L1111R/D1135V/G1218K/E1219V/A1322R/R1335Q mutation;
L1111R/D1135V/G1218Q/E1219Q/A1322R/R1333P/T1337L mutation;
L1111R/D1135V/G1218R/E1219F/A1322R/R1333G/R1335H/T1337C mutation; and
L1111R/D1135V/G1218M/E1219T/A1322R/R1333P/R1335Y/T1337L mutation.

### Claim 20 (depends on 19)

The SpCas9 variant of claim 19, wherein the SpCas9 variant comprises the L1111R/D1135V/G1218K/E1219V/A1322R/R1335Q mutation,
wherein the SpCas9 variant comprises an amino acid sequence of SEQ ID NO: 3,
wherein the SpCas9 variant is capable of recognizing PAM sequence which is 5′-NGN-3′.

### Claim 21 (depends on 19)

The SpCas9 variant of claim 19, wherein the SpCas9 variant comprises the L1111R/D1135V/G1218Q/E1219Q/A1322R/R1333P/T1337L mutation,
wherein the SpCas9 variant comprises an amino acid sequence of SEQ ID NO: 4,
wherein the SpCas9 variant is capable of recognizing PAM sequence which is 5′-NNG-3′.

### Claim 22 (depends on 19)

The SpCas9 variant of claim 19, wherein the SpCas9 variant comprises the L1111R/D1135V/G1218R/E1219F/A1322R/R1333G/R1335H/T1337C mutation,
wherein the SpCas9 variant comprises an amino acid sequence of SEQ ID NO: 5,
wherein the SpCas9 variant is PAMless.

### Claim 23 (depends on 19)

The SpCas9 variant of claim 19, wherein the SpCas9 variant comprises the L1111R/D1135V/G1218M/E1219T/A1322R/R1333P/R1335Y/T1337L mutation,
wherein the SpCas9 variant comprises an amino acid sequence of SEQ ID NO: 6,
wherein the SpCas9 variant is PAMless.

### Claim 24 (depends on 19)

A composition, comprising:
the SpCas9 variant of claim 19 or a nucleic acid encoding the SpCas9 variant; and
a guide RNA or a nucleic acid encoding the guide RNA,
wherein the guide RNA comprises a crRNA and a tracrRNA,
wherein the guide RNA is capable of forming a complex by interacting with the SpCas9 variant,
wherein the guide RNA is capable of binding to a target sequence of a target gene.

### Claim 25 (depends on 24)

The composition of claim 24, wherein the SpCas9 variant comprises an amino acid sequence of SEQ ID NO: 3,
wherein the SpCas9 variant is capable of recognizing PAM sequence which is 5′-NGN-3′.

### Claim 26 (depends on 24)

The composition of claim 24, wherein the SpCas9 variant comprises an amino acid sequence of SEQ ID NO: 4,
wherein the SpCas9 variant is capable of recognizing PAM sequence which is 5′-NNG-3′.

### Claim 27 (depends on 24)

The composition of claim 24, wherein the SpCas9 variant comprises an amino acid sequence of SEQ ID NO: 5,
wherein the SpCas9 variant is PAMless.

### Claim 28 (depends on 24)

The composition of claim 24, wherein the SpCas9 variant comprises an amino acid sequence of SEQ ID NO: 6,
wherein the SpCas9 variant is PAMless.

### Claim 29 (depends on 24)

The composition of claim 24, wherein the crRNA comprises a guide domain and a direct repeat, wherein a sequence of the direct repeat comprises at least 90% complementary to SEQ ID NO: 7,
wherein a sequence of the tracrRNA comprises at least 90% complementary to SEQ ID NO: 8.

### Claim 30 (depends on 24)

The composition of claim 24, wherein the composition comprises the SpCas9 variant and the guide RNA,
wherein the SpCas9 variant and the guide RNA are in a form of ribonucleoprotein (RNP).

### Claim 31 (depends on 24)

The composition of claim 24, wherein the composition comprises vector which comprises the nucleic acid encoding the SpCas9 variant and/or the nucleic acid encoding the guide RNA.

### Claim 32 (depends on 24)

The composition of claim 24, wherein the composition further comprises a Nuclear Localization Sequence (NLS) or a nucleic acid encoding the NLS.