# US 9,752,140 B2 · Methods and compostions for targeted genomic deletion

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- Number: US 9,752,140 B2
- Document ID: US9752140B2
- Status: Granted
- Kind: B2
- Application number: 15/007,569
- Filed: 2016-01-27
- Published: 2016-06-23
- Granted: 2017-09-05
- Earliest claimed: 2003-08-08
- Assignees: Sangamo Therapeutics, Inc.
- Applicants: Sangamo BioSciences, Inc.
- Inventors: Fyodor Urnov; Jianbin Wang; Michael C. Holmes
- Web page: https://patentgrep.com/patent/US9752140B2

## CPC

- C12N 15/102: {Mutagenizing nucleic acids}
- C12N 15/8213: {Targeted insertion of genes into the plant genome by homologous recombination}
- C12N 15/907: {in mammalian 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

Disclosed herein are compositions and methods for generating chromosomal translocations and targeted deletions of specific lengths and at specific locations the genome of cell.

## Claims

### Claim 1

A method for creating a targeted deletion of specific length and specific borders in a region of interest in a genomic locus in a host cell, the method comprising
introducing first and second nucleases and a donor polynucleotide into the host cell, wherein
(i) the first and second nucleases each comprise a 5′DNA-binding domain and a 3′ DNA binding domain and two catalytic domains;
(ii) the first nuclease binds to target sequences 5 to the target sequences in the region of interest bound by the second nuclease;
(iii) the 5′ DNA-binding domain binds to a target sequence 5′ to the target sequence bound by the 3′ DNA-binding domain,
(iv); the first nuclease binds to target sequences 5′ to the target sequences in the region of interest and the second nuclease binds to target sequences 3′ in the region of interest;
(v) the donor polynucleotide comprises the target sequences for the 5′ DNA binding domain of the first nuclease and target sequences for the 3′ DNA binding domain of the second nuclease and regions of homology to the genomic locus and further wherein the donor polynucleotide includes a deletion relative to the target sequence of specific length and specific borders, wherein the deletion extends between the target sequence 5′ DNA binding domain in of the first nuclease and target sequence for the 3′ DNA binding domain of the second nuclease; and
(vi) the donor polynucleotide is introduced into the genomic locus such that a targeted deletion of specific length and specific borders is generated in the genomic locus.

### Claim 2 (depends on 1)

The method of claim 1, wherein the regions of homology are between about 50 and 1500 base pairs in length.

### Claim 3 (depends on 1)

The method of claim 1, wherein the first nuclease cleaves at a first location and the second nuclease cleaves at a second location.

### Claim 4 (depends on 1)

The method of claim 1, wherein between the regions of homology, the donor polynucleotide comprises a sequence selected from the group consisting of a coding sequence, a 2A peptide, an SA site, an internal ribosome entry site (IRES), a shRNA molecule, an miRNA molecule, an RNAi and combinations thereof.

### Claim 5 (depends on 1)

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

### Claim 6 (depends on 5)

The method of claim 5, wherein the eukaryotic cell is a mammalian or plant cell.