Nucleotide-specific recognition sequences for designer tal effectors

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.

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.