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 .- 12 . (canceled) 
     
     
         13 . A TAL effector sequence containing a series of TAL nucleic acid binding cassettes selected from one or more of at least four different categories of cassettes encoding TAL repeats with all cassettes of one category binding to at least one of the bases adenine, guanine, thymidine, and cytosine in a nucleic acid target sequence, wherein the nucleotide composition of at least one first cassette in the series of cassettes differs from the nucleotide composition of all other cassettes of the same category. 
     
     
         14 . A TAL effector sequence according to  claim 13 , wherein the nucleotide composition of the at least one first cassette differs within a region of the cassette that is homologous in all other cassettes of said TAL effector sequence. 
     
     
         15 . A TAL effector sequence according to  claim 14 , wherein said homologous region has a length of at least 10, at least 15, or between 18 and 30 nucleotides. 
     
     
         16 . A TAL effector sequence according to  claim 14 , wherein the nucleotide composition of the at least one first cassette and/or the at least one second cassette differs within said homologous region by at least 3, preferably at least 4 nucleotides. 
     
     
         17 .- 21 . (canceled) 
     
     
         22 . A TAL effector fusion containing a TAL effector sequence according to  claim 13 , wherein the TAL effector fusion is a TAL-nuclease fusion. 
     
     
         24 . A vector containing a TAL effector sequence according to  claim 13 . 
     
     
         25 . (canceled) 
     
     
         26 . A method of sequencing a TAL effector sequence according to  claim 13 , wherein said method comprises using at least one sequencing primer specifically binding to the at least one first cassette within the TAL effector sequence. 
     
     
         27 .- 29 . (canceled) 
     
     
         30 . A method of detecting and identifying one or more genomic locus modifications comprising the steps of:
 a) isolating genomic DNA from (i) a cell treated with a TAL effector nuclease or a pair of TAL effector nucleases and (ii) an untreated cell,   b) cleaving the isolated genomic DNA obtained from both samples with a mixture of restriction enzymes,   c) mixing the samples containing cleaved DNA fragments,   d) subjecting the double stranded DNA fragments to a melting an re-hybridizing procedure,   e) ligating the ends of the re-hybridized DNA fragments with a first double-stranded DNA adapter containing at least one restrictions enzyme cleavage site at its 5′-end,   f) optionally purifying the adapter containing DNA fragments,   g) treating the DNA fragments containing the first adapter with a mismatch cleaving enzyme thereby obtaining a pool of cleaved and uncleaved DNA fragments,   h) ligating the ends of the cleaved and uncleaved DNA fragments with a second double-stranded DNA adapter lacking the restriction enzyme cleavage site of the first adapter,   i) treating the population of DNA fragments containing the second adapter with a restriction enzyme specifically cleaving the at least one restriction enzyme cleavage site at the 5′-end of the first adapter resulting in the release of the second adapter,   j) optionally separating the population of DNA fragments containing only the first adapter from the population of DNA fragments containing a first and a second adapter,   k) subjecting at least the population of DNA fragments containing a first and a second adapter to a sequencing reaction using the second adapter as primer binding site, and   l) identifying the one or more genomic locus modifications.   
     
     
         31 . A method according to  claim 30  wherein the mixture of restriction enzymes in step b) contains one or more restriction enzymes having a four or six base pair recognition sequence. 
     
     
         32 .- 36 . (canceled) 
     
     
         37 . A method for generating a population of product cells, the method comprising:
 (a) expressing the TAL-nuclease fusion of claim  23  in a population of starting cells to generate a sub-population of product cells that have undergone genetic recombination at a locus containing a detectable marker or selectable marker, wherein the TAL nuclease fusion is designed to bind to and cleave at least two nucleic acid loci in the population of starting cells and wherein at least one of the nucleic acid loci encodes the detectable marker or selectable marker, and   (b) generating the population of product cells by separating the product cells from the population of starting cells or selecting for the product cells.   
     
     
         38 .- 46 . (canceled) 
     
     
         47 . A method for the intracellular remodeling of chromatin, the method comprising expressing a TAL-chromatin modifier fusion in a cell, wherein the TAL nuclease fusion is designed to bind to a nucleic acid locus in the cell and modify the chromatin at the binding locus. 
     
     
         48 . The method of  claim 47 , wherein the chromatin modifier is a protein having at least one of ATPase, methylase, demethylase, acetylase, or deacetylase activities. 
     
     
         49 .- 51 . (canceled) 
     
     
         52 . 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:   
       
         
           
                 
                 
                 
                 
               
                     
                    (1) 
                   FSQADIVKIAGN, 
                   (SEQ ID NO: 37) 
                 
                     
                     
                 
                     
                    (2) 
                   GGAQALQAVLDLEP, 
                   (SEQ ID NO: 38) 
                 
                     
                     
                 
                     
                    (3) 
                   GGAQALQAVLDLEPALRERG, 
                   (SEQ ID NO: 39) 
                 
                     
                     
                 
                     
                    (4) 
                   FRTEDIVQMVS, 
                   (SEQ ID NO: 40) 
                 
                     
                     
                 
                     
                    (5) 
                   GGSKNLAAVQA, 
                   (SEQ ID NO: 41) 
                 
                     
                     
                 
                     
                    (6) 
                   GGSKNLEAVQA, 
                   (SEQ ID NO: 42) 
                 
                     
                     
                 
                     
                    (7) 
                   LEPKDIVSIAS, 
                   (SEQ ID NO: 43) 
                 
                     
                     
                 
                     
                    (8) 
                   GATQAITTLLNKW, 
                   (SEQ ID NO: 44) 
                 
                     
                     
                 
                     
                    (9) 
                   GATQAITTLLNKWDXLRAKG, 
                   (SEQ ID NO: 45) 
                 
                     
                   and 
                     
                     
                 
                     
                     
                 
                     
                   (10) 
                   GATQAITTLLNKWGXLRAKG; 
                   (SEQ ID NO: 46) 
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
         wherein X is one of the following amino acids: aspartic acid, serine, alanine, or glutamic acid. 
       
     
     
         53 . The non-naturally occurring protein of  claim 52 , 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  Xanthomonas  or  Ralstonia.    
     
     
         54 .- 60 . (canceled)