Nucleotide-specific recognition sequences for designer TAL effectors

1. 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′-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, 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 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 at least one or more KRAB domains or a fragment thereof.

2. The method according to claim 1 , wherein

the N-terminal capping region or fragment thereof comprises 147 contiguous amino acids of a wild type N-terminal capping region, or

the C-terminal capping region or fragment thereof comprises 68 contiguous amino acids of a wild type C-terminal capping region, or

the N-terminal capping region or fragment thereof comprises 136 contiguous amino acids of a wild type N-terminal capping region and the C-terminal capping region or fragment thereof comprises 183 contiguous amino acids of a wild type C-terminal capping region.

3. The method according to claim 1 , wherein at least one RVD is selected from the group consisting of (a) HH, KH, NH, NK, NQ, RH, RN, SS for recognition of guanine (G); (b) SI for recognition of adenine (A); (c) HG, KG, RG for recognition of thymine (T); (d) RD, SD 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.

4. The method according to claim 3 , wherein

the RVD for the recognition of G is RN, NH, RH or KH; or

the RVD for the recognition of A is SI; or

the RVD for the recognition of T is KG or RG; and

the RVD for the recognition of C is SD or RD.

5. The method according to claim 1 , wherein at least one of the following is present

[LTLD] (SEQ ID NO: 1) or [LTLA] (SEQ ID NO: 2) or [LTQV] (SEQ ID NO: 3) at X 1-4 , or

[EQHG] (SEQ ID NO: 4) or [RDHG] (SEQ ID NO: 5) at positions X 30-33 or X 31-34 or X 32-35 ,

wherein the sequence X 1-11 -X 12 X 13 -X 14-33 or 34 or 35 is selected from the group consisting of:

(SEQ ID NO: 356)

LTLDQVVAIAS X 12 X 13  GGKQALETVQRLLPVLCQDHG,

(SEQ ID NO: 357)

LTLDQVVAIAS X 12 X 13  GGKQALETVQRLLPVLCQAHG,

(SEQ ID NO: 358)

LTLDQVVAIAS X 12 X 13  GSKQALETVQRLLPVLCQDHG,

(SEQ ID NO: 359)

LTLDQVVAIAS X 12 X 13  GGKKALETVQRLLPVLCQDHG,

(SEQ ID NO: 360)

LTLDKVVAIAS X 12 X 13  GGKQALETVQRLLPVLCQDHG,

(SEQ ID NO: 361)

LTLDQVVAIAS X 12 X 13  GSKQALETVQRLLPVLCQAHG,

(SEQ ID NO: 362)

LTLDQVVAIAS X 12 X 13  GGKQALETVQRLLPVLCQARG,

(SEQ ID NO: 363)

LTLDQVVAIAS X 12 X 13  GGKQALETVQRLLPVLCEQHG,

(SEQ ID NO: 364)

LTLDQVVAIAS X 12 X 13  GGKQALETVQRLLPVLCQTHG,

(SEQ ID NO: 365)

LTLDQVAAIAS X 12 X 13  GGKQALETVQRLLPVLCQAHG,

(SEQ ID NO: 366)

LTLDQVVAIAS X 12 X 13  GSKQALETVQRLLPVLCQTHG,

(SEQ ID NO: 367)

LTLDQVVAIAS X 12 X 13  GGKQALETVQRLLPVLCQEHG,

(SEQ ID NO: 368)

LTLDQVVSIAS X 12 X 13  GGKQALETVQRLLPVLCQDHG,

(SEQ ID NO: 369)

LTLAQVVAIAS X 12 X 13  GGKQALETVQRLLPVLCQAHG,

(SEQ ID NO: 370)

LTLAQVVAIAS X 12 X 13  GGKQALETVQRLLPVLCQDHG,

(SEQ ID NO: 371)

LTLAQVVAIAN X 1 2X 13  GGKQALETVQRLLPVLCQAHG,

(SEQ ID NO: 372)

LTLAQVVAIAN X 12 X 13  GGKQALETVQRLLPVLCQDHG,

(SEQ ID NO: 373)

LTLAQVVAIAS X 12 X 13  GGKQALETVQRLLPVLCQTHG,

(SEQ ID NO: 374)

LTQVQVVAIAS X 12 X 13  GGKQALETVQRLLPVLCQAHG,

(SEQ ID NO: 376)

LTPDQVVAIAS X 12 X 13  GGKQALETVQRLLPVLCEQHG,

(SEQ ID NO: 377)

LTPAQVVAIAS X 12 X 13  GGKQALETVQQLLPVLCEQHG,

(SEQ ID NO: 378)

LTPAQVVAIAS X 12 X 13  GGKQALETVQRLLPVLCEQHG,

(SEQ ID NO: 379)

LTPDQVVAIAS X 12 X 13  GGRPALETVQRLLPVLCEQHG,

(SEQ ID NO: 380)

LTPAQVVAIAS X 12 X 13  GGKQALKTVQQLLPVLCEQHG,

(SEQ ID NO: 381)

LTPDQVVAIAS X 12 X 13  GGKQALERVQRLLPVLCEQHG,

(SEQ ID NO: 382)

LTRAQVVAIAS X 12 X 13  GGKQALETVQRLLPVLCEQHG,

(SEQ ID NO: 383)

LTLDQVVAIAS X 12 X 13  GGKQALETVQRLLPVLCEQHG,

(SEQ ID NO: 384)

LTPAQVVTIAS X 12 X 13  GGKQALETVQRLLPVLCEQHG,

(SEQ ID NO: 385)

LTPQQVVAIAS X 12 X 13  GGKQALETVQRLLPVLCEQHG,

(SEQ ID NO: 386)

LTPAQVVAIAS X 12 X 13  GGKPALETVQRLLPVLCEQHG,

(SEQ ID NO: 387)

LTPDQVVAIAS X 12 X 13  GGRQALETVQRLLPVLCEQHG,

(SEQ ID NO: 388)

LTPDQVVAIAS X 12 X 13  GGKPALETVQRLLPVLCEQHG,

(SEQ ID NO: 389)

LTPDQVVAIAS X 12 X 13  GGKQALETVQRLLPVLCRDHG,

and

(SEQ ID NO: 390)

LTPAQVVAIAS X 12 X 13  GGKQALETVQRLLPVLCRDHG.

6. The method according to claim 1 , wherein the sequence X 1-11 -X 12 X 13 -X 14-33 or 34 or 35 is LTLTQVVAIAS X 12 X 13 GGKQALETVQRLLPVLCQAHG (SEQ ID NO: 375).

7. The method according to claim 1 , wherein KRAB domain or a fragment thereof has a sequence:

(SEQ ID NO: 344)

MDAKSLTAWSRTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNL

VSLGYQLTKPDVILRLEKGEEPWLVEREIHQETHPDSETAFEIKSSV.

8. The method according to claim 1 , wherein KRAB domain or a fragment thereof has a sequence:

(SEQ ID NO: 345)

MDAKSLTAWSRTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNL

VSLGYQLTKPDVILRLEKGEEPWLV.

9. The method according to claim 1 , wherein KRAB domain or a fragment thereof has a sequence:

(SEQ ID NO: 346)

RTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGYQLTKP

DVILRLEKGEEPWLV.