# US 11,441,135 B2 · Target-specific CRISPR mutant

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

- Number: US 11,441,135 B2
- Document ID: US11441135B2
- Status: Granted
- Kind: B2
- Application number: 16/628,199
- Filed: 2018-07-06
- Published: 2020-05-14
- Granted: 2022-09-13
- Earliest claimed: 2017-07-07
- Assignees: Toolgen Incorporated
- Applicants: Toolgen Incorporated
- Inventors: Young-Hoon Kim; Min Hee Jung; Joonsun Lee; Eunji Shin; Kang In Lee; Seokjoong Kim; Jeongjoon Lee
- Web page: https://patentgrep.com/patent/US11441135B2

## CPC

- C12N 9/226: {Class 2 CAS enzyme complex, e.g. single CAS protein}
- C12N 15/102: {Mutagenizing nucleic acids}
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 9/78: acting on carbon to nitrogen bonds other than peptide bonds (3.5)
- C12N 15/86: Viral vectors
- C07K 2319/00: Fusion polypeptide
- C07K 2319/01: containing a localisation/targetting motif
- 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 invention relates to an artificially engineered CRISPR/Cas9 system. More particularly, the present invention relates to an artificially engineered CRISPR enzyme having enhanced target specificity and a use of an artificially engineered CRISPR/Cas9 system including the same enzyme in genome and/or epigenome manipulation or modification, genome targeting, genome editing, and in vitro diagnosis, etc.

## Claims

### Claim 1

A SpCas9 (Streptococcus pyogenes Cas9) variant comprising all of SEQ ID NO: 1 except for (i) a serine at the position corresponding to position 539 of the polypeptide of SEQ ID NO: 1; (ii) an isoleucine at the position corresponding to position 763 of the polypeptide of SEQ ID NO: 1; and (iii) an asparagine at the position corresponding to position 890 of the polypeptide of SEQ ID NO: 1, and wherein the SpCas9 variant has an endonuclease activity.

### Claim 2 (depends on 1)

The SpCas9 variant of claim 1, wherein the SpCas9 variant further comprises one or more functional domains selected from the group consisting of:
a tag for isolation and purification;
a deaminase; and
an activity domain having methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity, or nucleic acid binding activity.

### Claim 3 (depends on 2)

The SpCas9 variant of claim 2, wherein the SpCas9 variant further comprises a reporter protein, a NLS (nuclear localization sequence or signal), or a NES (nuclear export sequence or signal).

### Claim 4

A composition for gene modification comprising a Cas9 and a guide RNA, wherein the Cas9 is a Streptococcus pyogenes Cas9 (SpCas9) variant comprising all of SEQ ID NO: 1 except for (i) a serine at the position corresponding to position 539 of the polypeptide of SEQ ID NO: 1; (ii) an isoleucine at the position corresponding to position 763 of the polypeptide of SEQ ID NO: 1; and (iii) an asparagine at the position corresponding to position 890 of the polypeptide of SEQ ID NO: 1, and wherein the guide RNA is complementary to a target gene.

### Claim 5 (depends on 4)

The composition of claim 4, wherein the guide RNA and the Cas9 form a complex which is a ribonucleoprotein (RNP).

### Claim 6 (depends on 4)

The composition of claim 4, wherein the composition further comprises a donor nucleic acid that comprises a gene of interest.