Tal-effector assembly platform, customized services, kits and assays

Abstract

The invention generally relates to compositions and methods for designing and producing functional DNA binding effector molecules and associated customized services, tool kits and functional assays. In some aspects, the invention provides methods and tools for efficient assembly of customized TAL effector molecules. Furthermore, the invention relates to uses of TAL effector molecules and functional evaluation of such TAL by, for example, customized assays.

1 . A linear nucleic acid molecule comprising:
 (a) a region encoding an N-terminal portion of a TAL effector,   (b) a region encoding a C-terminal portion of a TAL effector,   (c) at least one recombination site, and   (d) at least one covalently bound topoisomerase,   wherein the topoisomerase is located at one of the termini of the linear nucleic acid molecule and is within 100 nucleotides of the at least one recombination site, and   wherein, when the nucleic acid molecule is circularized and contains a TAL repeat located between the termini of the nucleic acid molecule, the circularized nucleic acid molecule encode a TAL effector which is capable binding to a specified nucleic acid sequence.   
     
     
         2 . The linear nucleic acid molecule of  claim 1 , wherein the linear nucleic acid molecule contains an origin of replication. 
     
     
         3 . The linear nucleic acid molecule of  claim 1 , wherein the at least one recombination site is selected from the group consisting of:
 (a) an att site,   (b) a lox site, and   (c) a frt site.   
     
     
         4 . The linear nucleic acid molecule of  claim 1 , wherein the at least one covalently bound topoisomerase is a Type IA, Type IB, Type IIA, or Type II topoisomerase. 
     
     
         5 . (canceled) 
     
     
         6 . A method for preparing a TAL effector library, the method comprising:
 (a) connecting a population of TAL nucleic acid binding cassettes that individually encode adenine, guanine, thymidine, or cytosine base binders, when the base is present in a nucleic acid molecule, and   (b) introducing the connected TAL nucleic acid binding cassettes generated in (a) into a vector to generate a TAL effector library,   wherein the library encodes TAL effectors which bind to different nucleotide sequences.   
     
     
         7 . The method of  claim 6 , wherein TAL nucleic acid binding cassettes that encode adenine, guanine, thymidine, and cytosine binders are not all present in equimolar amounts. 
     
     
         8 . The method of  claim 6 , wherein TAL nucleic acid binding cassettes that encode adenine and thymine binders are present in equimolar amounts and represent from about 51% to about 75% of the total TAL nucleic acid binding cassettes present. 
     
     
         9 . The method of  claim 6 , wherein the TAL effector library encodes TAL effector fusions. 
     
     
         10 . The method of  claim 6 , wherein the TAL effector fusion have transcriptional activation activity. 
     
     
         11 . The method of  claim 6 , wherein the TAL effector fusions inhibit transcription. 
     
     
         12 . The method of  claim 6 , wherein the vector contains at least one recombination site. 
     
     
         13 . (canceled) 
     
     
         14 . A TAL effector library prepared by the method of  claim 6 . 
     
     
         15 . A method for identifying TAL effectors that bind to specified nucleotide sequences, the method comprising:
 (a) connecting a population TAL nucleic acid binding cassettes which individually encode TAL subunits that bind to one of the bases adenine, guanine, thymidine, and cytosine, when the base is present in a nucleic acid molecule,   (b) introducing the connected TAL nucleic acid binding cassettes generated in (a) into a vector to generate a TAL effector library, wherein the library contains TAL effectors which bind to different nucleotide sequences,   (c) introducing the TAL effector library into a cell under conditions which allow for the expression of TAL effectors, and   (d) screening the cells generated in (c) to identify cells in which at least one cellular parameter is altered by expression of a TAL effector.   
     
     
         16 . The method of  claim 15 , wherein the cellular parameter is TAL effector induced transcriptional activation of a non-TAL effector gene. 
     
     
         17 . The method of  claim 15 , wherein the cell contains nucleic acid comprising a promoter operably linked to a reporter and wherein the cellular parameter is transcriptional activation of the reporter. 
     
     
         18 . The method of  claim 17 , wherein the reporter is green fluorescent protein. 
     
     
         19 . The method of  claim 15 , wherein a TAL effector library member is isolated from a cell in which at least one cellular parameter is altered by expression of a TAL effector. 
     
     
         20 . A composition comprising a nucleic acid molecule encoding the TAL effector isolated by the method of  claim 15 . 
     
     
         21 . A non-naturally occurring protein comprising:
 (a) an amine terminal region of between 25 and 500 amino acids,   (b) a carboxyl terminal region of between 25 and 500 amino acids, and   (c) a central region containing five or more amino acid segments which confer upon the non-naturally occurring protein sequence specific nucleic acid binding activity,   wherein each of the individual amino acid segments in (c) are between 30 and 38 amino acid in length, and   wherein at least one of the amino acid segments is at least 80% identical to one or more of the following amino acid sequences:   
       
         
           
                 
                 
               
                     
                   (SEQ ID NO: 37) 
                 
                     
                   (1) FSQADIVKIAGN, 
                 
                     
                     
                 
                     
                   (SEQ ID NO: 38) 
                 
                     
                   (2) GGAQALQAVLDLEP, 
                 
                     
                     
                 
                     
                   (SEQ ID NO: 39) 
                 
                     
                   (3) GGAQALQAVLDLEPALRERG, 
                 
                     
                     
                 
                     
                   (SEQ ID NO: 40) 
                 
                     
                   (4) FRTEDIVQMVS, 
                 
                     
                     
                 
                     
                   (SEQ ID NO: 41) 
                 
                     
                   (5) GGSKNLAAVQA, 
                 
                     
                     
                 
                     
                   (SEQ ID NO: 42) 
                 
                     
                   (6) GGSKNLEAVQA, 
                 
                     
                     
                 
                     
                   (SEQ ID NO: 43) 
                 
                     
                   (7) LEPKDIVSIAS, 
                 
                     
                     
                 
                     
                   (SEQ ID NO: 44) 
                 
                     
                   (8) GATQAITTLLNKW, 
                 
                     
                     
                 
                     
                   (SEQ ID NO: 45) 
                 
                     
                   (9) GATQAITTLLNKWDXLRAKG, 
                 
                     
                   and 
                 
                     
                     
                 
                     
                   (SEQ ID NO: 46) 
                 
                     
                   (10) GATQAITTLLNKWGXLRAKG; 
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
         wherein X is one of the following amino acids: aspartic acid, serine, alanine, or glutamic acid. 
       
     
     
         22 . The non-naturally occurring protein of  claim 21 , wherein none of the amino acid segments is identical to an amino acid sequence of a TAL protein which naturally occurs in a bacterium of the genera  Xanthamonas  or  Ralstonia.    
     
     
         23 . The non-naturally occurring protein of  claim 21 , wherein at least one of the amino acid segments is not identical to an amino acid sequence shown in  FIG. 30 . 
     
     
         24 . The non-naturally occurring protein of  claim 21 , wherein the protein is a fusion protein. 
     
     
         25 . The non-naturally occurring fusion protein of  claim 24 , wherein the fusion protein comprises a sequence specific nucleic acid binding activity and at least a second activity other than sequence specific nucleic acid binding activity. 
     
     
         26 . A nucleic acid molecule comprising a sequence encoding the non-naturally occurring protein of  claim 21 . 
     
     
         27 . A vector comprising the nucleic acid molecule of  claim 26 . 
     
     
         28 . A host cell comprising the nucleic acid molecule of  claim 26 .