Nucleotide-Specific Recognition Sequences For Designer TAL Effectors

• US 20130137161A1
• Filed: 12/31/2012
• Published: 05/30/2013
• Est. Priority Date: 11/30/2011
• Status: Abandoned Application

• 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
• Application Number:
  US13/732,287
• Publication Number:
  US 20130137161A1
• Time in Patent Office: Days
• US Class Current: 435/194
• 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

1-16. -16. (canceled)

17. 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 five or more Transcription activator-like effector (TALE) monomers and at least one or more half-monomers specifically ordered to target a 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 at least one or more effector domains,

wherein the polypeptide is encoded by and translated from a codon optimized nucleic acid molecule so that the polypeptide preferentially binds to DNA of the genomic locus,

wherein the DNA binding domain comprises (X_1-11-X₁₂X₁₃-X_{14-33 or 34 or 35})_z,

wherein X_1-11 is a chain of 11 contiguous amino acids,

wherein X₁₂X₁₃ is a repeat variable diresidue (RVD),

wherein X_{14-33 or 34 or 35} is a chain of 21, 22 or 23 contiguous amino acids,

wherein z is at least 5 to 40, and

wherein at least one RVD is selected from the group consisting of HH, RD, SD, H*, N* and S* wherein (*) means that the amino acid at X₁₃ is absent.

18. The composition according to claim 17, wherein the at least one RVD for the recognition of cytosine (C) is selected from the group consisting of HH, RD and SD.

19. The composition according to claim 18, wherein the RVD for the recognition of C is SD.

20. The composition according to claim 17, wherein the wherein the genomic locus is in an animal cell.

21. The composition according to claim 20, wherein the animal is a mammal.

22. The composition according to claim 17, wherein the effector domain is an activator domain, a repressor domain, a DNA methyltransferase domain, a recombinase domain or a nuclease domain.

23. The composition according to claim 17, wherein at least one (X_1-11-X_14-34) or (X_1-11-X_14-35) is selected from FIG. 24.

24. The composition according to claim 17, 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.