Methods of Transcription Activator Like Effector Assembly

Abstract

The disclosure describes methods that include providing a first nucleic acid having a sequence encoding a first set comprising one or more transcription activator-like effector (TALE) repeat domains and/or one or more portions of one or more TALE repeat domains; contacting the first nucleic acid with a first enzyme, wherein the first enzyme creates a first ligatable end; providing a second nucleic acid having a sequence encoding a second set comprising one or more TALE repeat domains and/or one or more portions of one or more TALE repeat domains; contacting the second nucleic acid with a second enzyme, wherein the second enzyme creates a second ligatable end, and wherein the first and second ligatable ends are compatible; and ligating the first and second nucleic acids through the first and second ligatable ends to produce a first ligated nucleic acid, wherein the first ligated nucleic acid is linked to a solid support, and wherein the first ligated nucleic acid encodes a polypeptide comprising said first and second sets.

1 . A process comprising:
 (a) providing a first nucleic acid comprising a sequence encoding a first set comprising one or more transcription activator-like effector (TALE) repeat domains and/or one or more portions of one or more TALE repeat domains;   (b) contacting the first nucleic acid with a first enzyme, wherein the first enzyme creates a first ligatable end;   (c) providing a second nucleic acid comprising a sequence encoding a second set comprising one or more TALE repeat domains and/or one or more portions of one or more TALE repeat domains;   (d) contacting the second nucleic acid with a second enzyme, wherein the second enzyme creates a second ligatable end, and wherein the first and second ligatable ends are compatible; and   (e) ligating the first and second nucleic acids through the first and second ligatable ends to produce a first ligated nucleic acid,   wherein the first ligated nucleic acid is linked to a solid support, and   wherein the first ligated nucleic acid encodes a polypeptide comprising said first and second sets.   
     
     
         2 . The process of  claim 1 , wherein the first set is N-terminal to the second set in the polypeptide. 
     
     
         3 . The process of  claim 1 , wherein the second set is N-terminal to the first set in the polypeptide. 
     
     
         4 . The process of  claim 1 , wherein the first and second enzymes are a first and second restriction endonuclease, wherein the first restriction endonuclease cleaves at a site within the first nucleic acid and creates a first cut end, and the second restriction endonuclease cleaves at a site within the second nucleic acid and creates a second cut end, and wherein the first and second ligatable ends are the first and second cut ends. 
     
     
         5 . The process of  claim 4 , wherein the first ligated nucleic acid does not comprise a restriction site recognized by the first restriction endonuclease. 
     
     
         6 . The process of  claim 1 , further comprising:
 (f) contacting the first ligated nucleic acid with a third enzyme, wherein the third enzyme creates a third ligatable end;   (g) providing a third nucleic acid comprising a sequence encoding a third set comprising one or more TALE repeat domains and/or one or more portions of one or more TALE repeat domains;   (h) contacting the third nucleic acid with a fourth enzyme, wherein the fourth enzyme creates a fourth ligatable end, and wherein the third and fourth ligatable ends are compatible; and   (i) ligating the first ligated and third nucleic acids through the third and fourth ligatable ends to produce a second ligated nucleic acid linked to the solid support,   wherein the second ligated nucleic acid encodes a polypeptide comprising said first, second, and third sets.   
     
     
         7 . The process of  claim 6 , wherein the third and fourth enzymes are a third and fourth restriction endonuclease, wherein the third restriction endonuclease cleaves at a site within the first ligated nucleic acid and creates a third cut end, and the fourth restriction endonuclease cleaves at a site within the third nucleic acid and creates a fourth cut end, and wherein the third and fourth ligatable ends are the third and fourth cut ends. 
     
     
         8 . The process of  claim 7 , wherein the ligated nucleic acid does not comprise a restriction site recognized by the first endonuclease, and wherein the first and third restriction endonucleases are the same. 
     
     
         9 . The process of  claim 7 , wherein the second and fourth restriction endonucleases are the same. 
     
     
         10 . The process of  claim 6 , further comprising:
 (j) contacting the second ligated nucleic acid with a fifth enzyme, wherein the fifth enzyme creates a fifth ligatable end;   (k) providing a fourth nucleic acid comprising a sequence encoding a fourth set comprising one or more TALE repeat domains and/or one or more portions of one or more TALE repeat domains;   (l) contacting the fourth nucleic acid with a sixth enzyme, wherein the sixth enzyme creates a sixth ligatable end, and wherein the fifth and sixth ligatable ends are compatible; and   (m) ligating the second ligated and fourth nucleic acids through the fifth and sixth ligatable ends to produce a third ligated nucleic acid linked to the solid support, wherein the third ligated nucleic acid encodes a polypeptide comprising said first, second, third, and fourth sets.   
     
     
         11 . The process of  claim 10 , wherein the fifth and sixth enzymes are a fifth and sixth restriction endonuclease, wherein the fifth restriction endonuclease cleaves at a site within the second ligated nucleic acid and creates a fifth cut end, and the sixth restriction endonuclease cleaves at a site within the fourth nucleic acid and creates a sixth cut end, and wherein the fifth and sixth ligatable ends are the fifth and sixth cut ends. 
     
     
         12 . The process of  claim 11 , wherein the second ligated nucleic acid does not comprise a restriction site recognized by the first endonuclease, and wherein the first, third, and fifth restriction endonucleases are the same. 
     
     
         13 . The process of  claim 11 , wherein the second, fourth, and sixth restriction endonucleases are the same. 
     
     
         14 . The process of  claim 1 , wherein the second set comprises one to four TALE repeat domains. 
     
     
         15 . The process of  claim 1 , wherein the first and second ligatable ends each comprise an overhang of 1-10 nucleotides. 
     
     
         16 . The process of  claim 1 , wherein the first enzyme is a type IIS restriction endonuclease. 
     
     
         17 . The process of  claim 1 , further comprising unlinking the first ligated nucleic acid from the solid support and inserting the first ligated nucleic acid into a vector. 
     
     
         18 . The process of  claim 6 , further comprising unlinking the second ligated nucleic acid from the solid support and inserting the second ligated nucleic acid into a vector. 
     
     
         19 . The process of  claim 10 , further comprising unlinking the third ligated nucleic acid from the solid support and inserting the third ligated nucleic acid into a vector. 
     
     
         20 . The process of  claim 17 , wherein the vector is an expression vector. 
     
     
         21 . The process of  claim 20 , wherein the expression vector includes a sequence encoding an effector domain, and wherein the first, second, or third ligated nucleic acid is inserted into the vector such that the vector comprises a sequence encoding a fusion protein of the polypeptide and the effector domain. 
     
     
         22 . The process of  claim 21 , wherein the effector domain is a nuclease domain. 
     
     
         23 . The process of  claim 20 , further comprising inserting the expression vector into a cell. 
     
     
         24 . The process of  claim 23 , further comprising expressing the polypeptide or fusion protein. 
     
     
         25 . The process of  claim 24 , further comprising purifying the polypeptide or fusion protein. 
     
     
         26 .- 33 . (canceled)