# US 2025/0154531 A1 · Methods for improved homologous recombination and compositions thereof

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- Number: US 2025/0154531 A1
- Document ID: US20250154531A1
- Status: Published application
- Kind: A1
- Application number: 18/924,629
- Filed: 2024-10-23
- Published: 2025-05-15
- Earliest claimed: 2017-09-08
- Assignees: Life Technologies Corporation
- Applicants: Life Technologies Corporation
- Inventors: Chi-Hsuan CHANG; Xiquan LIANG
- Web page: https://patentgrep.com/patent/US20250154531A1

## CPC

- C12N 15/63: Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N 15/87: Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N 5/0636: {T lymphocytes}
- C12N 9/226: {Class 2 CAS enzyme complex, e.g. single CAS protein}
- C12N 15/11: DNA or RNA fragments; Modified forms thereof (DNA or RNA not used in recombinant technology, C07H21/00); {Non-coding nucleic acids having a biological activity}
- C12N 5/0634: {Cells from the blood or the immune system}
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N 2501/999: Small molecules not provided for elsewhere
- C12N 2510/00: Genetically modified cells
- C12N 2800/80: Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present disclosure relates to methods, kits, and compositions for improving the efficiency of homologous recombination. In particular, the disclosure relates to methods for introducing a nucleic acid cutting entity for DNA editing into cells that are difficult to transfect. Generally, the nucleic acid cutting entity is introduced into the cell without the use of viral vectors. The disclosure also relates to cloning DNA molecules directly into a genome with the combined use of promoter trapping and short homology arms, nuclear localization signal, and/or binding one or more DNA binding agents (TAL effector domain or truncated guide RNA bound by Cas9) to specific sites thereby displacing or restructuring chromatin at the target locus, and/or it increasing the accessibility of the target locus to further enzymatic modifications. The methods and compositions provided herein are, inter alia, useful for genome editing and enhancing enzymatic processes involved therein.

## Claims

### Claim 1

A method for genetically altering a cell, the method comprising:
(i) contacting a cell with a nucleic acid cutting entity and a donor DNA;
(ii) electroporating the cell under conditions that allow the nucleic acid cutting entity and the donor DNA to be taken up by the cell; and
(iii) culturing the cell in the presence of a non-homologous end joining (NHEJ) inhibitor, thereby forming a genetically altered cell;
wherein the cell is a stem cell, an immune cell, a primary cell, or a cell grown in suspension.

### Claim 2 (depends on 1)

The method of claim 1, wherein the nucleic acid cutting entity is a zinc finger protein, a transcription activator-like effector (TALE), a CRISPR complex, an argonaute-nucleic acid complex, a meganuclease or a macronuclease.

### Claim at position 3

-4. (canceled)

### Claim 5 (depends on 1)

The method of claim 1, wherein the cell is a primary cell.

### Claim 6 (depends on 1)

The method of claim 1, wherein the cell is a stem cell.

### Claim 7

(canceled)

### Claim 8 (depends on 1)

The method of claim 1, wherein the cell is an immune cell.

### Claim 9

(canceled)

### Claim 10 (depends on 8)

The method of claim 8, wherein the immune cell is a T cell.

### Claim 11 (depends on 1)

The method of claim 1, wherein the NHEJ inhibitor is a DNA-dependent protein kinase (DNA-PK) inhibitor, a DNA ligase IV inhibitor, or combination thereof.

### Claim 12

(canceled)

### Claim 13 (depends on 11)

The method of claim 11, wherein the DNA-PK inhibitor is Nu7026, Ku0060648 and/or Nu7441.

### Claim at position 13

-16. (canceled)

### Claim 17 (depends on 1)

The method of claim 1, wherein step (ii) is performed using an electroporation buffer and the NHEJ inhibitor is added to the electroporation buffer prior to step (ii).

### Claim 18

(canceled)

### Claim 19 (depends on 1)

The method of claim 1, wherein the cell is cultured in a cell culture media prior to step (i), and said cell culture media comprises the NHEJ inhibitor.

### Claim at position 17

-24. (canceled)

### Claim 25

A kit comprising:
(i) a non-homologous end joining (NHEJ) inhibitor; and
(ii) an electroporation buffer.

### Claim 26

(canceled)

### Claim 27 (depends on 25)

The kit of claim 25, further comprising a donor DNA.

### Claim 28 (depends on 25)

The kit of claim 25, further comprising a cell culture media.

### Claim at position 22

-33. (canceled)

### Claim 34

A method for genetically altering a population of cells, the method comprising:
(i) contacting the population of cells with a nucleic acid cutting entity, a donor DNA and a buffer suitable for electroporation, wherein the buffer suitable for electroporation comprises a non-homologous end joining (NHEJ) inhibitor and wherein the NHEJ inhibitor comprises a DNA-dependent protein kinase (DNA-PK) inhibitor;
(ii) electroporating the population of cells under conditions that allow the nucleic acid cutting entity and the donor DNA to be taken up by the cell; and
(iii) culturing the population of cells in cell culture medium, thereby forming a genetically altered cell.

### Claim 35 (depends on 34)

The method of claim 34, wherein the nucleic acid cutting entity comprises a transcription activator-like effector (TALE).

### Claim 36 (depends on 34)

The method of claim 34, wherein the nucleic acid cutting entity comprises a CRISPR complex.

### Claim 37 (depends on 34)

The method of claim 34, wherein the cell is a stem cell.

### Claim 38 (depends on 34)

The method of claim 34, wherein the cell is an immune cell.

### Claim 39 (depends on 38)

The method of claim 38, wherein the immune cell is a T cell.

### Claim 40 (depends on 34)

The method of claim 34, wherein the DNA-PK inhibitor is Nu7026, Ku0060648 and/or Nu7441.