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 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 regionwherein (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′
-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, 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,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 at least one or more KRAB domains or a fragment thereof.
4 Assignments
0 Petitions
Accused Products
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.
-
Citations
9 Claims
-
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′
-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, 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,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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9)
-
Specification