Nucleotide-specific recognition sequences for designer TAL effectors

A method of selectively targeting a genomic locus of interest comprising a coding or regulatory sequence susceptible to being affected by an effector domain in an animal cell, comprising contacting the genomic locus with a non-naturally occurring or engineered composition comprising a 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 at least one or more effector domains,
• 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,
• 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 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 [LTQV] (SEQ ID NO:
• 3) is present at X14, and
• 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) 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,
• or
• wherein [RDHG] (SEQ ID NO:
• 5) is present at positions X30-33 or X31-34 or X32-35, and
• 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) 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, RA, S* for recognition of A or T or G or C, wherein (*) means that the amino acid at X13 is absent,
• and
• wherein selectively targeting the genomic locus comprises specific recognition between the target DNA sequence and the polypeptide corresponding to the genomic locus of interest in the animal cell contacted with the polypeptide and an effect on the genomic locus of interest by the effector domain, as compared to a control animal cell in which the genomic locus of interest is contacted with a control polypeptide that does not have at least one or more effector domains.

A method of selectively targeting a genomic locus of interest comprising a coding or regulatory sequence susceptible to being affected by an effector domain in an animal cell, comprising contacting the genomic locus with a non-naturally occurring or engineered composition comprising a 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 at least one or more effector domains,
• 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,
• 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 the sequence X1-11-X12X13-X14-33 or 34 or 35 is LTLTQVVAIAS X12X13 GGKQALETVQRLLPVLCQAHG (SEQ ID NO:
• 375),
• 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) 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, and
• wherein selectively targeting the genomic locus comprises specific recognition between the target DNA sequence and the polypeptide corresponding to the genomic locus of interest in the animal cell contacted with the polypeptide and an effect on the genomic locus of interest by the effector domain, as compared to a control animal cell in which the genomic locus of interest is contacted with a control polypeptide that does not have at least one or more effector domains.