# US 2025/0146003 A1 · Compositions for Inducing Modifications of Target Endogenous Nucleic Acid Sequences in Nucleuses of Eukaryotic Cells

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- Number: US 2025/0146003 A1
- Document ID: US20250146003A1
- Status: Published application
- Kind: A1
- Application number: 19/015,836
- Filed: 2025-01-10
- Published: 2025-05-08
- Earliest claimed: 2013-06-20
- Assignees: Toolgen Incorporated
- Applicants: Toolgen Incorporated
- Inventors: Jin-Soo Kim; Seung Woo Cho; Sojung Kim
- Web page: https://patentgrep.com/patent/US20250146003A1

## CPC

- C12N 15/52: Genes encoding for enzymes or proenzymes
- A01K 67/0275: Genetically modified vertebrates, e.g. transgenic
- 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 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)
- 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)
- A01K: ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- 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 herein is a system for inducing targeted disruption of endogenous genes in a eukaryotic cell, the system comprising: an extracellular Cas9/RNA complex; cell-free and a buffer; wherein the extracellular Cas9/RNA complex comprises: a recombinant Cas9; and a guide RNA having a CRISPR RNA (crRNA) and a transactivating crRNA (tracrRNA); wherein the extracellular Cas9/RNA complex is disposed in the cell-free buffer and wherein the extracellular Cas9/RNA complex is complexed prior to being introduced into the eukaryotic cell; and wherein the Cas9/RNA complex functions as an endonuclease that induces targeted disruption of the target DNA upon introduction into the eukaryotic cell.

## Claims

### Claim 1

A system for inducing targeted disruption of endogenous genes in a eukaryotic cell, the system comprising:
an extracellular Cas9/RNA complex; and
a cell-free buffer;
wherein the extracellular Cas9/RNA complex comprises:
a recombinant Cas9; and
a guide RNA having a CRISPR RNA (crRNA) and a transactivating crRNA (tracrRNA);
wherein the extracellular Cas9/RNA complex is disposed in the cell-free buffer and
wherein the extracellular Cas9/RNA complex is complexed prior to being introduced into the eukaryotic cell; and
wherein the Cas9/RNA complex functions as an endonuclease that induces targeted disruption of the target DNA upon introduction into the eukaryotic cell.