Methods of transcription activator like effector assembly

Patent metadata

Field	Value
Publication number	US-10273271-B2
Application number	US-201615156574-A
Country	US
Kind code	B2
Filing date	May 17, 2016
Priority date	Jul 15, 2011
Publication date	Apr 30, 2019
Grant date	Apr 30, 2019

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.

First claim

What is claimed is:

1. A process comprising:
(a) providing a first nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 58-62;
(b) contacting the first nucleic acid with BsaI, thereby creating a ligatable end;
(c) providing a second nucleic acid comprising βγδε, wherein the second nucleic acid is constructed by:
(i) contacting a β unit comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 63-67 with BsaI to create a ligatable end, and contacting a γ unit comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 68-72 with BbsI to create a ligatable end
(ii) ligating the β unit and the γ unit through their ligatable ends to produce βγ;
(iii) contacting βγ with BsaI to create a ligatable end, and contacting a δ unit comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 73-77 with BbsI to create a ligatable end;
(iv) ligating βγ and the δ unit through their ligatable ends to produce βγδ;
(v) contacting βγδ with BsaI to create a ligatable end, and contacting a ε unit comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 78-82 with BbsI to create a ligatable end;
(vi) ligating βγδ and the ε unit through their ligatable ends to produce the second nucleic acid comprising βγδε;
(d) contacting the second nucleic acid with BbsI, thereby creating a ligatable end;
(e) ligating the first and second nucleic acids through their ligatable ends to produce a first ligated nucleic acid, and
(f) linking the first ligated nucleic acid to a solid support to produce a ligated nucleic acid linked to the solid support.

2. The process of claim 1 , further comprising:
(g) contacting the ligated nucleic acid linked to the solid support with BsaI, thereby creating a ligatable end;
(h) providing a third nucleic acid comprising βγδε, constructed as set forth in step (c) above;
(i) contacting the third nucleic acid with BbsI to create a ligatable end; and
(j) ligating the ligated nucleic acid linked to the solid support to the third nucleic acid through their ligatable ends to extend the ligated nucleic acid linked to the solid support, and
(k) optionally repeating steps (g)-(j).

3. The process of claim 2 , further comprising:
(l) contacting the ligated nucleic acid linked to the solid support with BsaI, thereby creating a ligatable end;
(m) providing a fourth nucleic acid, wherein the fourth nucleic acid is selected from one of the following nucleic acids:
i. a nucleic acid comprising a β unit,
ii. a nucleic acid comprising βγ′, wherein the nucleic acid is constructed by:
(a) contacting a β unit comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 63-67 with BsaI to create a ligatable end, and contacting a γ′ unit comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 83-87 with BbsI to create a ligatable end;
(b) ligating the β unit and the γ′ unit through their ligatable ends to produce βγ′; or
iii. a nucleic acid comprising βγδ, wherein the nucleic acid is constructed by:
(a) contacting a β unit comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 63-67 with BsaI to create a ligatable end, and contacting a γ unit comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 68-72 with BbsI to create a ligatable end;
(b) ligating the β unit and the γ unit through their ligatable ends to produce βγ;
(c) contacting βγ with BsaI to create a ligatable end, and contacting a δ unit comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 73-77 with BbsI to create a ligatable end;
(d) ligating βγ and the δ unit through their ligatable ends to produce βγδ;
(n) contacting the fourth nucleic acid with BbsI to create a ligatable end; and
(o) ligating the ligated nucleic acid linked to the solid support to the fourth nucleic acid through their ligatable ends to extend the ligated nucleic acid linked to the solid support.

4. The process of claim 2 , further comprising:
(l) contacting the ligated nucleic acid linked to the solid support with BsaI, thereby creating a ligatable end;
(m) providing a fourth nucleic acid comprising βγ, wherein the fourth nucleic acid is constructed by:
(i) contacting a β unit comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 63-67 with BsaI to create a ligatable end, and contacting a γ unit comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 68-72 with BbsI to create a ligatable end
(ii) ligating the β unit and the γ unit through their ligatable ends to produce βγ;
(n) contacting the fourth nucleic acid with BbsI, thereby creating a ligatable end; and
(o) ligating the ligated nucleic acid linked to the solid support to the fourth nucleic acid through their ligatable ends to extend the ligated nucleic acid linked to the solid support.

5. The process of claim 4 , further comprising:
(p) contacting the ligated nucleic acid linked to the solid support with BsaI, thereby creating a ligatable end;
(q) providing a fifth nucleic acid comprising a 6E′, wherein the fifth nucleic acid is constructed by:
(i) contacting a δ unit comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 73-77 with BsaI to create a ligatable end, and contacting an ε′ unit comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 88-92 with BbsI to create a ligatable end;
(ii) ligating the δ unit and the ε′ unit through their ligatable ends to produce δε′;
(r) contacting the fifth nucleic acid with BbsI, thereby creating an eighth ligatable end; and
(s) ligating the ligated nucleic acid linked to the solid support to the fifth nucleic acid through their ligatable ends to extend the ligated nucleic acid linked to the solid support.

6. The process of claim 1 , further comprising unlinking the ligated nucleic acid from the solid support and inserting said ligated nucleic acid into a vector.

7. The process of claim 2 , further comprising unlinking the ligated nucleic acid from the solid support and inserting said ligated nucleic acid into a vector.

8. The process of claim 3 , further comprising unlinking the ligated nucleic acid from the solid support and inserting said ligated nucleic acid into a vector.

9. The process of claim 6 , wherein the vector is an expression vector.

10. The process of claim 9 , wherein the expression vector includes a sequence encoding an effector domain, and wherein the ligated nucleic acid is inserted into the vector such that the vector comprises a sequence encoding a fusion protein of a TALE repeat domain and the effector domain.

11. The process of claim 10 , wherein the effector domain is a nuclease domain.

12. The process of claim 10 , further comprising inserting the expression vector into a cell.

13. The process of claim 12 , further comprising expressing the fusion protein.

14. The process of claim 13 , further comprising purifying the fusion protein.

Assignees

• Massachusetts Gen Hospital

Inventors

• Joung J Keith
• Sander Jeffry D

Classifications

• C12P19/34

  Polynucleotides, e.g. nucleic acids, oligoribonucleotides · CPC title

• C07K2319/41

  containing a Myc-tag · CPC title

• C07K2319/43

  containing a FLAG-tag · CPC title

• C07K2319/00

  Fusion polypeptide · CPC title

• C12Q1/6806

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title