Nucleotide-specific recognition sequences for designer TAL effectors
View Patent ↗The invention relates to methods of altering expression of a genomic locus of interest or specifically targeting a genomic locus of interest in an animal cell, which may involve contacting the genomic locus with a non-naturally occurring or engineered composition that includes a deoxyribonucleic acid (DNA) binding polypeptide having a N-terminal capping region, a DNA binding domain comprising at least five or more Transcription activator-like effector (TALE) monomers and at least one or more half-monomers specifically ordered to target the genomic locus of interest, and a C-terminal capping region, wherein the polypeptide includes at least one or more effector domains, and wherein the polypeptide is encoded by and translated from a codon optimized nucleic acid molecule so that the polypeptide preferentially binds to the DNA of the genomic locus.
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.
2. 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.
3. The method according to claim 2 , 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.
4. 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:
LTLDQVVAIAS X 12 X 13 GGKQALETVQRLLPVLCQDHG (SEQ ID NO: 356),
LTLDQVVAIAS X 12 X 13 GGKQALETVQRLLPVLCQAHG (SEQ ID NO: 357),
LTLDQVVAIAS X 12 X 13 GSKQALETVQRLLPVLCQDHG (SEQ ID NO: 358),
LTLDQVVAIAS X 12 X 13 GGKKALETVQRLLPVLCQDHG (SEQ ID NO: 359),
LTLDKVVAIAS X 12 X 13 GGKQALETVQRLLPVLCQDHG (SEQ ID NO: 360),
LTLDQVVAIAS X 12 X 13 GSKQALETVQRLLPVLCQAHG (SEQ ID NO: 361),
LTLDQVVAIAS X 12 X 13 GGKQALETVQRLLPVLCQARG (SEQ ID NO: 362),
LTLDQVVAIAS X 12 X 13 GGKQALETVQRLLPVLCEQHG (SEQ ID NO: 363),
LTLDQVVAIAS X 12 X 13 GGKQALETVQRLLPVLCQTHG (SEQ ID NO: 364),
LTLDQVAAIAS X 12 X 13 GGKQALETVQRLLPVLCQAHG (SEQ ID NO: 365),
LTLDQVVAIAS X 12 X 13 GSKQALETVQRLLPVLCQTHG (SEQ ID NO: 366),
LTLDQVVAIAS X 12 X 13 GGKQALETVQRLLPVLCQEHG (SEQ ID NO: 367),
LTLDQVVSIAS X 12 X 13 GGKQALETVQRLLPVLCQDHG (SEQ ID NO: 368),
LTLAQVVAIAS X 12 X 13 GGKQALETVQRLLPVLCQAHG (SEQ ID NO: 369),
LTLAQVVAIAS X 12 X 13 GGKQALETVQRLLPVLCQDHG (SEQ ID NO: 370),
LTLAQVVAIAN X 12 X 13 GGKQALETVQRLLPVLCQAHG (SEQ ID NO: 371),
LTLAQVVAIAN X 12 X 13 GGKQALETVQRLLPVLCQDHG (SEQ ID NO: 372),
LTLAQVVAIAS X 12 X 13 GGKQALETVQRLLPVLCQTHG (SEQ ID NO: 373),
LTQVQVVAIAS X 12 X 13 GGKQALETVQRLLPVLCQAHG (SEQ ID NO: 374),
LTPDQVVAIAS X 12 X 13 GGKQALETVQRLLPVLCEQHG (SEQ ID NO: 376),
LTPAQVVAIAS X 12 X 13 GGKQALETVQQLLPVLCEQHG (SEQ ID NO: 377),
LTPAQVVAIAS X 12 X 13 GGKQALETVQRLLPVLCEQHG (SEQ ID NO: 378),
LTPDQVVAIAS X 12 X 13 GGRPALETVQRLLPVLCEQHG (SEQ ID NO: 379),
LTPAQVVAIAS X 12 X 13 GGKQALKTVQQLLPVLCEQHG (SEQ ID NO: 380),
LTPDQVVAIAS X 12 X 13 GGKQALERVQRLLPVLCEQHG (SEQ ID NO: 381),
LTRAQVVAIAS X 12 X 13 GGKQALETVQRLLPVLCEQHG (SEQ ID NO: 382),
LTLDQVVAIAS X 12 X 13 GGKQALETVQRLLPVLCEQHG (SEQ ID NO: 383),
LTPAQVVTIAS X 12 X 13 GGKQALETVQRLLPVLCEQHG (SEQ ID NO: 384),
LTPQQVVAIAS X 12 X 13 GGKQALETVQRLLPVLCEQHG (SEQ ID NO: 385),
LTPAQVVAIAS X 12 X 13 GGKPALETVQRLLPVLCEQHG (SEQ ID NO: 386),
LTPDQVVAIAS X 12 X 13 GGRQALETVQRLLPVLCEQHG (SEQ ID NO: 387),
LTPDQVVAIAS X 12 X 13 GGKPALETVQRLLPVLCEQHG (SEQ ID NO: 388),
LTPDQVVAIAS X 12 X 13 GGKQALETVQRLLPVLCRDHG (SEQ ID NO: 389), and
LTPAQVVAIAS X 12 X 13 GGKQALETVQRLLPVLCRDHG (SEQ ID NO: 390).
5. 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.
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).