Method to overcome DNA chemical modifications sensitivity of engineered tale DNA binding domains

Patent metadata

Field	Value
Publication number	US-10301614-B2
Application number	US-201314387162-A
Country	US
Kind code	B2
Filing date	Mar 15, 2013
Priority date	Mar 23, 2012
Publication date	May 28, 2019
Grant date	May 28, 2019

Abstract

The present invention relates to polypeptides and more particularly to Transcription Activator-Like Effector derived proteins that allow to efficiently target and/or process nucleic acids. Particularly, the present invention reports the characterization of TALE derived proteins that can efficiently target methylated DNA. The present invention more specifically relates to TALE derived proteins that allow activation of methylated promoters responsible for gene silencing.

First claim

The invention claimed is:

1. A method to process a nucleic acid target sequence comprising a 5-methyl-cytosine comprising:
(a) providing cells containing a nucleic acid target sequence that comprises a 5-methyl-cytosine;
(b) introducing into said cell a polynucleotide comprising:
(i) a first polynucleotide encoding a transcription activator-like effector (TALE) protein comprising a plurality of TALE-like repeat sequences, each repeat comprising a repeat variable-diresidue (RVD) specific to each nucleic acid base of said nucleic acid target sequence, wherein the RVD that specifically targets the 5-methyl-cytosine within said nucleic acid target sequence is selected from N*, T*, Q* and H*, wherein * represents a gap in one position of the RVD; and
(ii) a second polynucleotide encoding an additional protein domain that has a nuclease activity, polymerase activity, kinase activity, phosphatase activity, methylase activity, topoisomerase activity, integrase activity, transposase activity, ligase activity, helicase activity or a recombinase activity;

(c) expressing said polynucleotide to form a chimeric protein that binds said nucleic acid target sequence and processes the nucleic acid within or adjacent to said nucleic acid target sequence and,
(d) selecting the cells in which said TALE protein has processed said nucleic acid target sequence within or adjacent to said nucleic acid target sequence;
wherein the TALE protein comprising an RVD N*, T*, Q* or H* that specifically targets the 5-methyl-cytosine can bind said nucleic acid target sequence more efficiently than a variant TALE protein having the RVD NG at the same position.

2. The method according to claim 1 , wherein said nucleic acid target sequence comprises at least one methylated CpG dinucleotide.

3. The method according to claim 1 , wherein said nucleic acid target sequence comprises at least one methylated CpA dinucleotide.

4. The method according to claim 1 , wherein said nucleic acid target sequence comprises at least one methylated CpT dinucleotide.

5. The method according to claim 1 , wherein said nucleic acid target sequence comprises at least one methylated CpC dinucleotide.

6. The method according to claim 1 , wherein the RVD that specifically targets the 5-methyl-cytosine within said nucleic acid target sequence is N*.

7. The method according to claim 1 , wherein the RVD that specifically targets the 5-methyl-cytosine within said nucleic acid target sequence is T*.

8. The method according to claim 1 , wherein the RVD that specifically targets the 5-methyl-cytosine within said nucleic acid target sequence is Q*.

9. The method according to claim 1 , wherein the RVD that specifically targets the 5-methyl-cytosine within said nucleic acid target sequence is H*.

10. The method according to claim 1 , wherein said nucleic acid target sequence is a methylated promoter sequence.

11. The method according to claim 6 , wherein said additional protein domain is an endonuclease.

12. The method according to claim 7 , wherein said additional protein domain is an endonuclease.

13. The method according to claim 8 , wherein said additional protein domain is an endonuclease.

14. The method according to claim 9 , wherein said additional protein domain is an endonuclease.

15. The method according to claim 1 , further comprising providing to the cell an exogenous nucleic acid comprising a sequence homologous to at least a portion of the nucleic acid target sequence, such that homologous recombination occurs between the nucleic acid target sequence and the exogenous nucleic acid.

16. The method according to claim 1 , wherein said chimeric protein is a TALE-nuclease that comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 39, 41, 44, 47, and 49.

Assignees

• Cellectis Sa
• Cellectis

Inventors

• Duchateau Philippe
• Valton Julien

Classifications

• C12N9/22

  Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} · CPC title

• C12N15/102 Primary

  Mutagenizing nucleic acids · CPC title

• C07K2319/80

  containing a DNA binding domain, e.g. Lacl or Tet-repressor · CPC title

• C07K2319/61

  containing an enzyme fusion for detection (lacZ, luciferase) · CPC title

• C07K14/47

  from mammals · CPC title