Modular dna-binding domains and methods of use

Abstract

The present invention refers to methods for selectively recognizing a base pair in a DNA sequence by a polypeptide, to modified polypeptides which specifically recognize one or more base pairs in a DNA sequence and, to DNA which is modified so that it can be specifically recognized by a polypeptide and to uses of the polypeptide and DNA in specific DNA targeting as well as to methods of modulating expression of target genes in a cell.

1 . A method for producing a polypeptide that selectively recognizes at least one base pair in a target DNA sequence, the method comprising synthesizing a polypeptide comprising a repeat domain, wherein the repeat domain comprises at least one repeat unit derived from a transcription activator-like (TAL) effector, wherein the repeat unit comprises a hypervariable region which determines recognition of a base pair in the target DNA sequence, wherein the repeat unit is responsible for the recognition of one base pair in the DNA sequence, and wherein the hypervariable region comprises a member selected from the group consisting of:
 (a) HD for recognition of C/G;   (b) NI for recognition of A/T;   (c) NG for recognition of T/A;   (d) NS for recognition of C/G or A/T or T/A or G/C;   (e) NN for recognition of G/C or A/T;   (f) IG for recognition of T/A;   (g) N for recognition of C/G or T/A;   (h) HG for recognition of C/G or T/A;   (i) H for recognition of T/A;   (j) NK for recognition of G/C;   (k) NH for recognition of G/C;   (l) NP for recognition of A/T or C/G or T/A;   (m) NT for recognition of A/T or G/C;   (n) HN for recognition of A/T or G/C;   (o) SH for recognition of G/C;   (p) SN for recognition of G/C; and   (q) IS for recognition of A/T;   
       wherein the repeat domain comprises at least one repeat unit which comprises a hypervariable region comprising (k), (l), (m), (n), (o), (p), or (q). 
     
     
         2 . (canceled) 
     
     
         3 . The method of  claim 1 , wherein the hypervariable region corresponds to amino acids 12 and 13 in the repeat unit. 
     
     
         4 . The method of  claim 1 , wherein the repeat domain comprises 1.5 to 40.5 repeat units. 
     
     
         5 . The method of  claim 1 , wherein the repeat domain comprises 11.5 to 33.5 repeat units. 
     
     
         6 . The method of  claim 1 , wherein the polypeptide further comprises at least one additional domain that is operably linked to the repeat domain. 
     
     
         7 . The method of  claim 6 , wherein the additional domain comprises a bacterial, viral, fungal, oomycete, human, animal, plant, or artificial protein, or part thereof. 
     
     
         8 . The method of  claim 6 , wherein the additional domain comprises a protein or functional part or domain thereof, that is capable of modifying DNA or RNA. 
     
     
         9 . The method of  claim 6 , wherein the additional domain comprises a protein or functional part or domain thereof selected from the group consisting of: a transcription activator, a transcription repressor, a resistance-mediating protein, a nuclease, a topoisomerase, a ligase, an integrase, a recombinase, a resolvase, a methylase, an acetylase, a demethylase, and a deacetylase. 
     
     
         10 . The method of  claim 1 , wherein the repeat domain of the polypeptide is synthesized by expressing a DNA sequence encoding the polypeptide and where the DNA sequence encoding the polypeptide is assembled by preassembling the repeat units in one or more target vectors that can subsequently be assembled into a final vector comprising the DNA sequence encoding the polypeptide. 
     
     
         11 . The method of  claim 1 , wherein the repeat unit comprises 30 to 40 amino acids. 
     
     
         12 . The method of  claim 11 , wherein the repeat unit comprises 33, 34, 35 or 39 amino acids. 
     
     
         13 . The method of  claim 1 , wherein the polypeptide recognizes at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 base pairs in the target DNA sequence. 
     
     
         14 . The method of  claim 13 , wherein the polypeptide recognizes all of the base pairs in the target DNA sequence. 
     
     
         15 . The method of  claim 13 , wherein the polypeptide is capable of binding to the target DNA sequence. 
     
     
         16 . A polypeptide produced by the method of  claim 1 . 
     
     
         17 . The polypeptide of  claim 16 , wherein the polypeptide is not naturally occurring. 
     
     
         18 . A polynucleotide molecule comprising a coding sequence for the polypeptide produced by the method of  claim 1 . 
     
     
         19 . The polynucleotide molecule of  claim 18 , wherein the polynucleotide molecule is not naturally occurring. 
     
     
         20 . An expression cassette comprising a promoter operably linked to the polynucleotide molecule of  claim 18 . 
     
     
         21 . A non-human host cell comprising the expression cassette of  claim 20 . 
     
     
         22 . The host cell of  claim 21 , wherein the host cell is a bacterial cell, a fungal cell, an animal cell, or a plant cell. 
     
     
         23 . A transformed, non-human organism comprising the expression cassette of  claim 20 . 
     
     
         24 . The transformed organism of  claim 23 , wherein the organism is a fungus, an animal, or a plant. 
     
     
         25 . A method for selectively recognizing a base pair in a DNA sequence by a polypeptide, the method comprising constructing a polypeptide comprising a repeat domain, wherein the repeat domain comprises at least one repeat unit derived from a TAL effector, wherein the repeat unit comprises a hypervariable region which determines recognition of a base pair in the DNA sequence, wherein the repeat unit is responsible for the recognition of one base pair in the DNA sequence, and wherein the hypervariable region comprises a member selected from the group consisting of:
 (a) HD for recognition of C/G;   (b) NI for recognition of A/T;   (c) NG for recognition of T/A;   (d) NS for recognition of C/G or A/T or T/A or G/C;   (e) NN for recognition of G/C or A/T;   (f) IG for recognition of T/A;   (g) N for recognition of C/G or T/A;   (h) HG for recognition of C/G or T/A;   (i) H for recognition of T/A;   (j) NK for recognition of G/C;   (k) NH for recognition of G/C;   (l) NP for recognition of A/T or C/G or T/A;   (m) NT for recognition of A/T or G/C;   (n) HN for recognition of A/T or G/C;   (o) SH for recognition of G/C;   (p) SN for recognition of G/C; and   (q) IS for recognition of A/T;   
       wherein the repeat domain comprises at least one repeat unit which comprises a hypervariable region comprising (k), (l), (m), (n), (o), (p), or (q). 
     
     
         26 . (canceled) 
     
     
         27 . The method of  claim 25 , wherein the hypervariable region corresponds to amino acids 12 and 13 in the repeat unit. 
     
     
         28 . The method of  claim 25 , wherein the repeat domain comprises 1.5 to 40.5 repeat units. 
     
     
         29 . The method of  claim 25 , wherein the repeat domain comprises 11.5 to 33.5 repeat units. 
     
     
         30 . The method of  claim 25 , wherein the polypeptide further comprises at one additional domain that is operably linked to the repeat domain. 
     
     
         31 . The method of  claim 25 , wherein the repeat unit comprises 30 to 40 amino acids. 
     
     
         32 . The method of  claim 31 , wherein the repeat unit comprises 33, 34, 35 or 39 amino acids. 
     
     
         33 . The method of  claim 25 , wherein the repeat domain comprising repeat units is inserted in a bacterial, viral, fungal, oomycete, human, animal or plant polypeptide to achieve a targeted recognition and preferably binding of one or more specified base pairs in a DNA sequence, and optionally wherein the repeat unit is derived from the repeat domains of AvrBs3-like effectors which are further optionally modified in order to obtain a pre-selected specific activity to one or more base pairs in a DNA sequence. 
     
     
         34 . The method of  claim 25 , wherein the repeat domain comprising the repeat unit is contained in a polypeptide controlling the transcription of a gene, optionally in transcription activator or repressor proteins, optionally in AvrBs3-like proteins, e.g. in AvrBs3 or Hax effector proteins. 
     
     
         35 . The method of  claim 25 , wherein the N-terminal region of a repeat domain confers a recognition specificity for a T/A 5′ of the recognition specificity of the repeat unit. 
     
     
         36 . The method of  claim 25 , wherein the base pair in the DNA sequence is inserted into an expression control element combined with a gene, the expression control element being targeted by a transcription control protein comprising the hypervariable region in the repeat unit recognizing the base pair located in the expression control element to specifically control the expression of the gene, wherein the expression control element is preferably a promoter. 
     
     
         37 . The method of  claim 36 , wherein the gene is a resistance mediating gene in order to obtain a disease resistant organism, the expression control element being optionally the target sequence for an AvrBs3-like effector protein. 
     
     
         38 . A method of modulating expression of a target gene in a cell, wherein cells are provided which contain a polypeptide wherein the polypeptide comprises a repeat domain, wherein the repeat domain comprises at least one repeat unit derived from a TAL effector, wherein the repeat unit comprises a hypervariable region which determines recognition of a base pair in a DNA sequence, wherein the repeat unit is responsible for the recognition of one base pair in the DNA sequence, and wherein the hypervariable region comprises a member selected from the group consisting of:
 (a) HD for recognition of C/G;   (b) NI for recognition of A/T;   (c) NG for recognition of T/A;   (d) NS for recognition of C/G or A/T or T/A or G/C;   (e) NN for recognition of G/C or A/T;   (f) IG for recognition of T/A;   (g) N for recognition of C/G or T/A;   (h) HG for recognition of C/G or T/A;   (i) H for recognition of T/A;   (j) NK for recognition of G/C;   (k) NH for recognition of G/C;   (l) NP for recognition of A/T or C/G or T/A;   (m) NT for recognition of A/T or G/C;   (n) HN for recognition of A/T or G/C;   (o) SH for recognition of G/C;   (p) SN for recognition of G/C; and   (q) IS for recognition of A/T;   
       wherein the repeat domain comprises at least one repeat unit which comprises a hypervariable region comprising (k), (l), (m), (n), (o), (p), or (q). 
     
     
         39 . (canceled) 
     
     
         40 . A polypeptide comprising a repeat domain, wherein the repeat domain comprises at least one repeat unit derived from a TAL effector, wherein the repeat unit comprises a hypervariable region which determines recognition of a base pair in a DNA sequence, wherein the repeat unit is responsible for the recognition of one base pair in the DNA sequence, and wherein the hypervariable region comprises a member selected from the group consisting of:
 (a) HD for recognition of C/G;   (b) NI for recognition of A/T;   (c) NG for recognition of T/A;   (d) NS for recognition of C/G or A/T or T/A or G/C;   (e) NN for recognition of G/C or A/T;   (f) IG for recognition of T/A;   (g) N for recognition of C/G or T/A;   (h) HG for recognition of C/G or T/A;   (i) H for recognition of T/A;   (j) NK for recognition of G/C;   (k) NH for recognition of G/C;   (l) NP for recognition of A/T or C/G or T/A;   (m) NT for recognition of A/T or G/C;   (n) HN for recognition of A/T or G/C;   (o) SH for recognition of G/C;   (p) SN for recognition of G/C; and   (q) IS for recognition of A/T;   
       wherein the repeat domain comprises at least one repeat unit which comprises a hypervariable region comprising (k), (l), (m), (n), (o), (p), or (q). 
     
     
         41 . (canceled) 
     
     
         42 . A polynucleotide molecule comprising a coding sequence for the polypeptide of  claim 40 . 
     
     
         43 . A DNA which is modified to include a at least one base pair located in a target DNA sequence so that the base pair can be specifically recognized by a polypeptide comprising a repeat domain, wherein the repeat domain comprises at least one repeat unit derived from a TAL effector, wherein the repeat unit comprises a hypervariable region which determines recognition of a base pair in the DNA sequence, wherein the repeat unit is responsible for the recognition of one base pair in the DNA sequence, and wherein the base pair is selected from the group consisting of:
 (a) G/C for recognition of NH;   (b) A/T or C/G or T/A for recognition of NP;   (c) A/T or G/C for recognition of NT;   (d) A/T or G/C for recognition of HN;   (e) G/C for recognition of SH;   (f) G/C for recognition of SN; and   (g) A/T for recognition of IS.   
     
     
         44 . The DNA of  claim 43 , wherein at least one additional base pair is selected from the group consisting of:
 (h) C/G for recognition by HD;   (i) A/T for recognition by NI;   (j) T/A for recognition by NG;   (k) CT or A/T or T/A or G/C for recognition by NS;   (l) G/C or A/T for recognition by NN;   (m) T/A for recognition by IG;   (n) C/G or T/A for recognition by N;   (o) T/A for recognition by HG;   (p) T/A for recognition by H; and   (q) G/C for recognition by NK.   
     
     
         45 . The DNA of  claim 43 , wherein the base pair is located in a promoter or other gene regulatory sequence. 
     
     
         46 . The DNA of  claim 43 , wherein the DNA is not naturally occurring. 
     
     
         47 . A vector comprising the DNA of  claim 43 . 
     
     
         48 . A non-human host cell comprising the DNA of  claim 43 . 
     
     
         49 . The host cell of  claim 43 , wherein the host cell is a bacterial cell, a fungal cell, an animal cell, or a plant cell. 
     
     
         50 . A transformed, non-human organism comprising the DNA of  claim 43 . 
     
     
         51 . The transformed organism of  claim 50 , wherein the organism is a fungus, an animal, or a plant. 
     
     
         52 . A method for producing a DNA comprising a target DNA sequence that is selectively recognized by a polypeptide comprising a repeat domain, wherein the repeat domain comprises at least one repeat unit derived from a TAL effector, wherein the repeat unit comprises a hypervariable region which determines recognition of a base pair in the target DNA sequence, wherein the repeat unit is responsible for the recognition of one base pair in the target DNA sequence, the method comprising synthesizing a DNA comprising a base pair that is capable of being recognized by the repeat unit, and wherein the base pair is selected from the group consisting of:
 (a) C/G for recognition by HD;   (b) A/T for recognition by NI;   (c) T/A for recognition by NG;   (d) CT or A/T or T/A or G/C for recognition by NS;   (e) G/C or A/T for recognition by NN;   (f) T/A for recognition by IG;   (g) C/G or T/A for recognition by N;   (h) T/A for recognition by HG;   (i) T/A for recognition by H;   (j) G/C for recognition by NK;   (k) G/C for recognition of NH;   (l) A/T or C/G or T/A for recognition of NP;   (m) A/T or G/C for recognition of NT;   (n) A/T or G/C for recognition of HN;   (o) G/C for recognition of SH;   (p) G/C for recognition of SN; and   (q) A/T for recognition of IS and   
       wherein the repeat domain comprises at least one repeat unit which comprises a hypervariable region comprising at least one member selected from the group consisting of
 (i) NH for recognition of G/C; 
 (ii) NP for recognition of A/T or C/G or T/A; 
 (iii) NT for recognition of A/T or G/C; 
 (iv) HN for recognition of A/T or G/C; 
 (v) SH for recognition of G/C; 
 (vi) SN for recognition of G/C; and 
 (vii) IS for recognition of A/T. 
 
     
     
         53 . (canceled)