# US 2025/0146002 A1 · Compositions for Inducing Modifications of Target Endogenous Nucleic Acid Sequences in Nucleuses of Eukaryotic Cells

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- Number: US 2025/0146002 A1
- Document ID: US20250146002A1
- Status: Published application
- Kind: A1
- Application number: 19/013,018
- Filed: 2025-01-08
- Published: 2025-05-08
- Earliest claimed: 2012-10-23
- Assignees: Toolgen Incorporated
- Applicants: Toolgen Incorporated
- Inventors: Jin-Soo Kim; Seung Woo Cho; Sojung Kim
- Web page: https://patentgrep.com/patent/US20250146002A1

## CPC

- C12N 15/52: Genes encoding for enzymes or proenzymes
- A61K 48/005: {characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered}
- C11D 3/386: Preparations containing enzymes {, e.g. protease or amylase}
- C11D 3/38663: {Stabilised liquid enzyme compositions}
- C11D 3/38681: {Chemically modified or immobilised enzymes}
- C11D 17/0073: {Tablets}
- C11D 17/045: {Multi-compartment}
- C12N 9/16: acting on ester bonds (3.1)
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 9/226: {Class 2 CAS enzyme complex, e.g. single CAS protein}
- C12N 9/54: bacteria being Bacillus
- 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 15/8216: {Methods for controlling, regulating or enhancing expression of transgenes in plant cells}
- C12N 15/85: for animal cells
- C12N 15/907: {in mammalian cells}
- C12Y 301/21: Endodeoxyribonucleases producing 5'-phosphomonoesters (3.1.21)
- C12Y 304/21062: Subtilisin (3.4.21.62)
- C12N 2310/10: Type of nucleic acid
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N 2310/531: Stem-loop; Hairpin
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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)
- C11D: DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C12Y: ENZYMES

## Abstract

The present disclosure relates to targeted genome editing in eukaryotic cells or organisms. More particularly, the present disclosure provides for compositions and methods that may induce modifications in target endogenous nucleic acid sequences in nucleuses of eukaryotic cells. For example, disclosed here in is a method of introducing a site-specific, double-stranded break at a target nucleic acid sequence in a eukaryotic cell, the method comprising introducing into the eukaryotic cell a Type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas system, wherein the CRISPR/Cas system comprises: a) a nucleic acid encoding a Cas9 polypeptide, wherein the Cas9 polypeptide comprises a nuclear localization signal, and b) a chimeric guide RNA comprising a CRISPR RNA (crRNA) portion fused to a trans activating crRNA (tracrRNA) portion, wherein the target nucleic acid sequence comprises a first strand having a region complementary to the crRNA portion of the chimeric guide RNA and a second strand having a trinucleotide protospacer adjacent motif (PAM), and whereby the site-specific, double-stranded break at the target nucleic acid sequence is introduced in the eukaryotic cell.

## Claims

### Claim 1

A method of introducing a site-specific, double-stranded break at a target nucleic acid sequence in a eukaryotic cell, the method comprising
introducing into the eukaryotic cell a Type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas system, wherein the CRISPR/Cas system comprises:
a) a nucleic acid encoding a Cas9 polypeptide, wherein the Cas9 polypeptide comprises a nuclear localization signal, and
b) a chimeric guide RNA comprising a CRISPR RNA (crRNA) portion fused to a trans activating crRNA (tracrRNA) portion, wherein the target nucleic acid sequence comprises a first strand having a region complementary to the crRNA portion of the chimeric guide RNA and a second strand having a trinucleotide protospacer adjacent motif (PAM), and
whereby the site-specific, double-stranded break at the target nucleic acid sequence is introduced in the eukaryotic cell.