Methods and compostions for targeted genomic deletion

• US 9,752,140 B2
• Filed: 01/27/2016
• Issued: 09/05/2017
• Est. Priority Date: 08/08/2003
• Status: Expired due to Term

• Current Assignee: Sangamo Therapeutics, Inc.
• Original Assignee: Sangamo Therapeutics, Inc.
• Inventors: Urnov, Fyodor, Wang, Jianbin, Holmes, Michael C.
• Primary Examiner(s): Leith, Nancy J
• Application Number:
  US15/007,569
• Publication Number:
  US 20160177290A1
• Time in Patent Office: 587 Days
• Field of Search: None
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12N 15/8213 Targeted insertion of genes into the plant genome by homologous recombination
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 2800/80 Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites

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

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.

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

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

4. 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.

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

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