Nucleotide-Specific Recognition Sequences For Designer TAL Effectors
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.
1 - 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 -X 12 X 13 -X 14-33 or 34 or 35 ) z ,
wherein X 1-11 is a chain of 11 contiguous amino acids,
wherein X 12 X 13 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 X 13 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.