Nucleotide-specific recognition sequences for designer TAL effectors

A method of repressing expression of a genomic locus of interest comprising a coding or regulatory sequence susceptible to repression by a KRAB repressor domain or a fragment thereof having activity of a KRAB repressor 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 genomic locus comprises a target DNA sequence 5′-T0N1N2 . . . Nz Nz+1-3′, where T0 and N=A, G, T or C,
• wherein the target DNA sequence binds to the DNA binding domain, and the DNA binding domain comprises (X1-11-X12X13-X14-33 or 34 or 35)z,
• wherein X1-11 is a chain of 11 contiguous amino acids,
• wherein X12X13 is a repeat variable diresidue (RVD),
• wherein X14-33 or 34 or 35 is a chain of 21, 22 or 23 contiguous amino acids,
• wherein z is at least 5 to 40,
• wherein at least one RVD is selected from the group consisting of (a) HH, KH, NH, NK, NQ, RH, RN, SS, NN, SN, KN for recognition of guanine (G);
• (b) NI, KI, RI, HI, SI for recognition of adenine (A);
• (c) NG, HG, KG, RG for recognition of thymine (T);
• (d) RD, SD, HD, ND, KD, YG 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,
• 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, and
• wherein repressing expression of the genomic locus comprises a decrease in transcript level corresponding to the genomic locus of interest in the mammalian cell contacted with the polypeptide as compared to a control mammalian cell in which the genomic locus of interest is contacted with a control polypeptide that does not have at least one or more KRAB domains or a fragment thereof.