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TALE (Transcription Activator Like Effectors) protein and application thereof

A protein and KG technology, applied in the field of genetic engineering, can solve the problems of reduced degree of freedom, low efficiency, RVD and DNA base specificity are not completely strict, etc., to improve specificity and efficiency, diversify selection, and reduce off-target effects. Effect

Inactive Publication Date: 2013-12-11
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, although the target sites of TALEs can be predicted according to the specificity of RVDs, many natural TALEs proteins contain RVDs that do not match the binding sites, indicating that the specificity of RVD binding to DNA bases is not completely strict.
The lack of RVD specificity limits the application of artificially synthesized TALE proteins, especially the NN used to bind the G base and also binds to the A base with a high affinity, making the selection of the target site in the TALE design The degree of freedom of the TALE protein is greatly reduced, the efficiency of TALE protein binding to the target site is affected, and the possibility of potential off-target effects increases, especially in applications related to therapy that have high requirements for off-target effects. strict TALE protein
[0004] In 2012, Jens Boch's group and Feng Zhang's group identified NH as the RVD that specifically recognizes G bases, but the efficiency of binding to G is lower than that of NN. In addition, NK also specifically binds to G, but the efficiency is lower (Streubel, J., C. Blucher, et al. (2012); Cong, L., R. Zhou, et al. (2012))

Method used

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  • TALE (Transcription Activator Like Effectors) protein and application thereof
  • TALE (Transcription Activator Like Effectors) protein and application thereof
  • TALE (Transcription Activator Like Effectors) protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 TALE transcriptional activator containing new RVD-KN

[0034] According to Table 1, KN is the RVD that specifically binds to the G base. For target site ACTC GGGGGGGGGGGG , Design a TALE-VP64 transcription activator with 15.5 repeats, and use RVD-----KN in the 5-15.5 repeats, and use the currently commonly used RVDs (NI-A, NI-A, NG-T, HD-C), the effect of activating the expression of the reporter gene downstream of the target site should be obtained. Since KN has specificity for G base binding, if the target site is replaced with ACTC AAAAAAAAAAAA , ACTC TTTTTTTTTTT T Or ACTC CCCCCCCCCCCC , Neither can activate the expression of downstream genes.

[0035] The constructed coding nucleotide sequence of the KN-containing TALE-VP64 transcription activator is shown in SEQ ID No. 1. The expression plasmid containing the KN TALE-VP64 gene (starting vector pLL3.7, purchased from Addgene, Inc.) and the reporter plasmid were co-transfected into the HEK293T cell line wi...

Embodiment 2

[0037] Example 2 TALE transcriptional activator containing novel RVD----RG

[0038] According to Table 1, RG is an RVD that specifically binds to the T base. For the target site CTGGCC TTT TACGTA, designed the TALE-VP64 transcription activator with 14.5 repeats, and used RVD in the seventh, eighth, and ninth repeats, and used the currently commonly used RVD (NI- A, NG-T, HD-C, NN-G), the effect of activating the downstream reporter gene expression of the target site should be obtained. Since RG has specific binding to T bases, if the target site is replaced with CTGGCC AAA TACGTA, CTGGCC CCC TACGTA or CTGGCC GGG TACGTA cannot activate the expression of downstream genes.

[0039] The coding nucleotide sequence of the constructed TALE-VP64 transcription activator containing RG is shown in SEQ ID No.2. The expression plasmid of the TALE-VP64 gene containing RG (starting vector pLL3.7) and the reporter plasmid were co-transfected into the HEK293T cell line with the transfection ...

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Abstract

The invention provides a novel TALE (Transcription Activator Like Effectors) protein and the application of the novel TALE protein. Through screening 400 different RVDs, a novel RVD of which the specificity is bound with a certain DNA basic group or combined with more than one DNA basic group at the same time is successfully identified, the joint efficiency of the novel RVD and the basic group or groups is detected, the novel RVD is used for synthesizing the TALE protein highly specifically and efficiently combined with a DNA target site, so that the choice of the target site of the TALE protein is more diversified, and the potential off-target effect is reduced.

Description

Technical field [0001] The invention relates to the field of genetic engineering, in particular to a new type of TALE protein and its application. Background technique [0002] TALEs (Transcription activator like effectors) are the third type of secreted proteins produced by a variety of Xanthomonas bacteria to activate host gene expression or promote self-communization (Boch and Bonas, 2010; Bogdanove et al. , 2010; Scholze and Boch, 2011). It has been reported that the amino acid sequence of the nucleic acid binding domain of TALEs protein has a relatively constant relationship with the nucleic acid sequence of its target site. The TALEs protein contains a highly conserved tandem repeat sequence composed of 33-35 amino acids as units, and each unit The 12th and 13th amino acid has variability, called RVD (repeat-variable diresidue), which is responsible for recognizing and specifically binding a single nucleotide (for example, NI recognizes A, HD recognizes C, NG recognizes T,...

Claims

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Application Information

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IPC IPC(8): C07K14/195C12N15/31C12N15/63C12N15/11C12N15/09
Inventor 魏文胜杨君娇张媛
Owner PEKING UNIV
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