Nucleotide-specific recognition sequences for designer TAL effectors
First Claim
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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Abstract
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.
62 Citations
6 Claims
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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:
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(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′
-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, 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. - View Dependent Claims (2, 3, 4, 5, 6)
the RVD for the recognition of A is SI;
orthe RVD for the recognition of T is KG or RG; and the RVD for the recognition of C is SD or RD.
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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 X1-4, or[EQHG] (SEQ ID NO;
4) or [RDHG] (SEQ ID NO;
5) at positions X30-33 or X31-34 or X32-35,wherein the sequence X1-11-X12X13-X14-33 or 34 or 35 is selected from the group consisting of; LTLDQVVAIAS X12X13 GGKQALETVQRLLPVLCQDHG (SEQ ID NO;
356),LTLDQVVAIAS X12X13 GGKQALETVQRLLPVLCQAHG (SEQ ID NO;
357),LTLDQVVAIAS X12X13 GSKQALETVQRLLPVLCQDHG (SEQ ID NO;
358),LTLDQVVAIAS X12X13 GGKKALETVQRLLPVLCQDHG (SEQ ID NO;
359),LTLDKVVAIAS X12X13 GGKQALETVQRLLPVLCQDHG (SEQ ID NO;
360),LTLDQVVAIAS X12X13 GSKQALETVQRLLPVLCQAHG (SEQ ID NO;
361),LTLDQVVAIAS X12X13 GGKQALETVQRLLPVLCQARG (SEQ ID NO;
362),LTLDQVVAIAS X12X13 GGKQALETVQRLLPVLCEQHG (SEQ ID NO;
363),LTLDQVVAIAS X12X13 GGKQALETVQRLLPVLCQTHG (SEQ ID NO;
364),LTLDQVAAIAS X12X13 GGKQALETVQRLLPVLCQAHG (SEQ ID NO;
365),LTLDQVVAIAS X12X13 GSKQALETVQRLLPVLCQTHG (SEQ ID NO;
366),LTLDQVVAIAS X12X13 GGKQALETVQRLLPVLCQEHG (SEQ ID NO;
367),LTLDQVVSIAS X12X13 GGKQALETVQRLLPVLCQDHG (SEQ ID NO;
368),LTLAQVVAIAS X12X13 GGKQALETVQRLLPVLCQAHG (SEQ ID NO;
369),LTLAQVVAIAS X12X13 GGKQALETVQRLLPVLCQDHG (SEQ ID NO;
370),LTLAQVVAIAN X12X13 GGKQALETVQRLLPVLCQAHG (SEQ ID NO;
371),LTLAQVVAIAN X12X13 GGKQALETVQRLLPVLCQDHG (SEQ ID NO;
372),LTLAQVVAIAS X12X13 GGKQALETVQRLLPVLCQTHG (SEQ ID NO;
373),LTQVQVVAIAS X12X13 GGKQALETVQRLLPVLCQAHG (SEQ ID NO;
374),LTPDQVVAIAS X12X13 GGKQALETVQRLLPVLCEQHG (SEQ ID NO;
376),LTPAQVVAIAS X12X13 GGKQALETVQQLLPVLCEQHG (SEQ ID NO;
377),LTPAQVVAIAS X12X13 GGKQALETVQRLLPVLCEQHG (SEQ ID NO;
378),LTPDQVVAIAS X12X13 GGRPALETVQRLLPVLCEQHG (SEQ ID NO;
379),LTPAQVVAIAS X12X13 GGKQALKTVQQLLPVLCEQHG (SEQ ID NO;
380),LTPDQVVAIAS X12X13 GGKQALERVQRLLPVLCEQHG (SEQ ID NO;
381),LTRAQVVAIAS X12X13 GGKQALETVQRLLPVLCEQHG (SEQ ID NO;
382),LTLDQVVAIAS X12X13 GGKQALETVQRLLPVLCEQHG (SEQ ID NO;
383),LTPAQVVTIAS X12X13 GGKQALETVQRLLPVLCEQHG (SEQ ID NO;
384),LTPQQVVAIAS X12X13 GGKQALETVQRLLPVLCEQHG (SEQ ID NO;
385),LTPAQVVAIAS X12X13 GGKPALETVQRLLPVLCEQHG (SEQ ID NO;
386),LTPDQVVAIAS X12X13 GGRQALETVQRLLPVLCEQHG (SEQ ID NO;
387),LTPDQVVAIAS X12X13 GGKPALETVQRLLPVLCEQHG (SEQ ID NO;
388),LTPDQVVAIAS X12X13 GGKQALETVQRLLPVLCRDHG (SEQ ID NO;
389), andLTPAQVVAIAS X12X13 GGKQALETVQRLLPVLCRDHG (SEQ ID NO;
390).
- 1) or [LTLA] (SEQ ID NO;
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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 X1-11-X12X13-X14-33 or 34 or 35 is LTLTQVVAIAS X12X13 GGKQALETVQRLLPVLCQAHG (SEQ ID NO:
- 375).
Specification