Patent ID: 8450107
Filing Date: 2013-05-28
Classification: C07K,C12N,C12Q

Abstract:
1. A method of repressing expression of a genomic locus of interest comprising a coding or regulatory sequence susceptible to repression by a SID4X repressor domain 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 polypeptide includes the SID4X repressor domain, and the SID4X repressor domain has the sequence MNIQMLLEAADYLERREREAEHGYASMLPGSGMNIQMLLEAADYLERREREAEHGYA SMLPGSGMNIQMLLEAADYLERREREAEHGYASMLPGSGMNIQMLLEAADYLERRER EAEHGYASMLPSR (SEQ ID NO: 50), wherein the genomic locus comprises a target DNA sequence 5′-T 0 N 1 N 2 . . . N z N z+1 -3′, where T 0 and N=A, G, T or C, wherein the target DNA sequence binds to the DNA binding domain, and 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, 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, HO, 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 X 13 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 the SID4X repressor domain.