NUCLEOTIDE-SPECIFIC RECOGNITION SEQUENCES FOR DESIGNER TAL EFFECTORS

• US 20130137174A1
• Filed: 09/06/2012
• Published: 05/30/2013
• Est. Priority Date: 11/30/2011
• Status: Active Grant

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation), Massachusetts Institute of Technology, The Broad Institute, Inc.
• Original Assignee: Massachusetts Institute of Technology, The Broad Institute, Inc.
• Inventors: Zhang, Feng, Cong, Le
• Granted Patent: US 8,450,107 B1
• Time in Patent Office: Days
• US Class Current: 435/375
• CPC Class Codes: C07K 14/03 Herpetoviridae, e.g. pseudorabies virus
• CPC Class Codes: C07K 14/195 from bacteria
• CPC Class Codes: C07K 14/4703 Inhibitors; Suppressors
• CPC Class Codes: C07K 2319/80 containing a DNA binding domain, e.g. Lacl or Tet-repressor
• CPC Class Codes: C12N 15/635 Externally inducible repressor mediated regulation of gene expression, e.g. tetR inducible by tetracyline
• CPC Class Codes: C12N 5/06 Animal cells or tissues; Human cells or tissues preservation of living cells or tissues A01N1/02
• CPC Class Codes: C12N 5/0686 Kidney cells
• CPC Class Codes: C12N 9/1241 Nucleotidyltransferases (2.7.7)
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2522/10 Nucleic acid binding proteins

Abstract

The invention relates to methods of altering expression of a genomic locus of interest or specifically targeting a genomic locus of interest in an animal cell, which may involve contacting the genomic locus with a non-naturally occurring or engineered composition that includes a deoxyribonucleic acid (DNA) binding polypeptide having a N-terminal capping region, a DNA binding domain comprising at least five or more Transcription activator-like effector (TALE) monomers and at least one or more half-monomers specifically ordered to target the genomic locus of interest, and a C-terminal capping region, wherein the polypeptide includes at least one or more effector domains, and wherein the polypeptide is encoded by and translated from a codon optimized nucleic acid molecule so that the polypeptide preferentially binds to the DNA of the genomic locus.

Claims

1-10. -10. (canceled)

11. A method of repressing expression of a genomic locus of interest in a mammalian cell, comprising contacting the genomic locus with a non-naturally occurring or engineered composition comprising a deoxyribonucleic acid (DNA) binding polypeptide comprising:

(a) a N-terminal capping region

(b) a DNA binding domain comprising at least 5 to 40 Transcription activator-like effector (TALE) monomers and at least one or more half-monomers specifically ordered to target the genomic locus of interest, and

(c) a C-terminal capping region

wherein (a), (b) and (c) are arranged in a predetermined N-terminus to C-terminus orientation,

wherein the polypeptide includes a SID4X repressor domain having the sequence

13. The method according to claim 11, wherein at least one RVD is selected from the group consisting of (a) HH, KH, NH, NK, NQ, RH, RN, SS for recognition of guanine (G);

(b) SI for recognition of adenine (A);

(c) HG, KG, RG for recognition of thymine (T);

(d) RD, SD for recognition of cytosine (C);

(e) NV, HN for recognition of A or G and (f) H*, HA, KA, N*, NA, NC, NS, RA, S*for recognition of A or T or G or C, wherein (*) means that the amino acid at X₁₃ is absent.

14. The method according to claim 13, wherein

the RVD for the recognition of G is RN, NH, RH or KH;

or

the RVD for the recognition of A is SI;

or

the RVD for the recognition of T is KG or RG; and

the RVD for the recognition of C is SD or RD.

15. The method according to claim 11, wherein at least one of the following is present

[LTLD] (SEQ ID NO:

1) or [LTLA] (SEQ ID NO;

     2) or [LTQV] (SEQ ID NO;

     3) at X_1-4, or

[EQHG] (SEQ ID NO;

     4) or [RDHG] (SEQ ID NO;

     5) at positions X_30-33 or X_31-34 or X_32-35,

wherein the sequence X_1-11-X₁₂X₁₃-X_{14-33 or 34 or 35} is selected from the group consisting of;

16. The method according to claim 11, wherein

the N-terminal capping region or fragment thereof comprises 147 contiguous amino acids of a wild type N-terminal capping region, or

the C-terminal capping region or fragment thereof comprises 68 contiguous amino acids of a wild type C-terminal capping region, or

the N-terminal capping region or fragment thereof comprises 136 contiguous amino acids of a wild type N-terminal capping region and the C-terminal capping region or fragment thereof comprises 183 contiguous amino acids of a wild type C-terminal capping region.

12. (canceled)