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 . A non-naturally occurring or engineered 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 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 effector domain having deaminase activity.
2 . The DNA binding polypeptide of claim 1 , wherein the locus is in an animal cell.
3 . The DNA binding polypeptide of claim 2 , wherein the animal is a mammal.
4 . The DNA binding polypeptide of claim 1 , wherein the DNA is single stranded DNA.
5 . The DNA binding polypeptide of claim 1 , wherein the DNA is double stranded DNA.
6 . The DNA binding polypeptide of claim 1 , wherein
the N-terminal capping region comprises 147 contiguous amino acids of a wild type N-terminal capping region, or
the C-terminal capping region comprises 68 contiguous amino acids of a wild type C-terminal capping region, or
the N-terminal capping region comprises 136 contiguous amino acids of a wild type N-terminal capping region and the C-terminal capping region comprises 183 contiguous amino acids of a wild type C-terminal capping region.
7 . A non-naturally occurring or engineered composition comprising a nucleic acid molecule encoding a deoxyribonucleic acid (DNA) binding polypeptide, wherein the DNA binding polypeptide comprises:
(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 the 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 effector domain having deaminase activity.
8 . The composition of claim 7 , further comprising an expression vector, wherein the nucleic acid molecule is cloned into the expression vector.
9 . A pharmaceutical composition comprising the polypeptide of claim 1 and optionally one or more pharmaceutically acceptable excipients.
10 . A method of selectively targeting a locus of interest comprising a coding or a regulatory sequence susceptible to being affected by an effector domain in a mammalian cell, comprising contacting the locus with 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 the 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 effector domain having deaminase activity.
11 . The method of claim 10 , further comprising delivering of a nucleic acid molecule encoding the polypeptide to the mammalian cell.
12 . The method of claim 10 , wherein the locus of interest is in an animal cell.
13 . The method of claim 10 , wherein the animal is a mammal.
14 . The DNA binding polypeptide of claim 10 , wherein the DNA is single stranded DNA.
15 . The DNA binding polypeptide of claim 10 , wherein the DNA is double stranded DNA.