Tale RVD specifically recognizing DNA base modified by methylation and application thereof

Patent metadata

Field	Value
Publication number	US-11897920-B2
Application number	US-201716636283-A
Country	US
Kind code	B2
Filing date	Aug 4, 2017
Priority date	Aug 4, 2017
Publication date	Feb 13, 2024
Grant date	Feb 13, 2024

Abstract

RVDs with recognition preferences for 5mC, 5hmC and 6 mA and different binding properties to these epigenetic modifications are identified in this present invention. Methylation-dependent gene activation, efficient genome editing, targeted detection of 5hmC and other applications can be achieved by using these RVDs. The present invention therefore provides an isolated DNA binding polypeptide containing TALEs, a fusion protein, a polynucleotide, a vector comprising the polynucleotide and a host cell, and the use of the protein comprising TALE repeats domain in the preparation of a reagent for detecting a methylated base in a target sequence of a gene of interest, as well as a method for targeting and binding to a target sequence of a gene of interest in a cell.

First claim

The invention claimed is:

1. A method for detecting the presence of 5-methylcytosine (5mC) at a specific site of a target sequence in the genome of a cell, comprising:
(1) introducing a protein comprising transcription activator-like effectors (TALEs) into the cell, wherein the TALEs target the target sequence, and in the TALEs the repeat variable diresidue (RVD) identifying the specific site is HA;
(2) introducing a nuclease into the cell, wherein the targeted cleavage site of the nuclease is located in the TALEs target sequence; and
(3) detecting whether the target sequence is cleaved, thereby judging whether 5mC is present at the specific site of the target sequence; if the target sequence is not cleaved, then the TALEs bind to the target sequence, and the nuclease does not bind to the target sequence and cleave it, thereby 5mC is present at the specific site; if the target sequence is cleaved, then the TALEs do not bind to the target sequence, and the nuclease binds to the target sequence and cleaves it, thereby 5mC is not present at the specific site.

2. The method of claim 1 , wherein the nuclease is an endonuclease.

3. The method of claim 2 , wherein the nuclease is a Cas9 nuclease.

4. The method of claim 3 , wherein the Cas9 nuclease and an sgRNA are co-introduced into the cell in step (2).

Assignees

• Univ Beijing
• Edigene Inc

Inventors

• Wei Wensheng
• Yi Chengqi
• Zhang Yuan
• Guo Shengjie
• Zhu Chenxu
• Liu Lulu

Classifications

• C07K14/195 Primary

  from bacteria · CPC title

• C12N9/22

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

• C12N15/113

  Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)} · CPC title

• C12N15/66

  General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease · CPC title

• C07K2319/00

  Fusion polypeptide · CPC title