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Nucleotide-specific recognition sequences for designer TAL effectors

  • US 8,450,107 B1
  • Filed: 09/06/2012
  • Issued: 05/28/2013
  • Est. Priority Date: 11/30/2011
  • Status: Active Grant
First Claim
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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 regionwherein (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′

    -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, 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 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, andwherein 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.

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