Chimeric nuclease for specific recognition and repair of beta thalassemia beta-globin gene

An amino acid and nucleic acid molecule technology, applied in the field of genetic engineering, which can solve problems such as obstacles and off-targets

Active Publication Date: 2016-06-29
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, ZFN can only recognize a small number of sites at present, and there will be relatively large limitations in application; TALEN technology has the characteristics of easy assembly, fast speed, and strong recognition, but because TALEN contains FokI, it has non-specificity in recognition, resulting in certain off-target; CRISPR / Cas9 system has the characteristics of simple operation, but its binding to nucleic acid is very non-specific, which will be an important obstacle to its application

Method used

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  • Chimeric nuclease for specific recognition and repair of beta thalassemia beta-globin gene
  • Chimeric nuclease for specific recognition and repair of beta thalassemia beta-globin gene
  • Chimeric nuclease for specific recognition and repair of beta thalassemia beta-globin gene

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Experimental program
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Effect test

Embodiment 1

[0037] Example 1. Preparation of a chimeric nuclease that specifically recognizes and repairs the beta-thalassemia beta-globin gene

[0038] 1. Transformation of homing endonuclease I-SceI

[0039] The homing endonuclease I-SceI will use a special fluorescent reporter system in human cells to screen the DNA sequence that specifically recognizes the DNA sequence adjacent to the β-thalassemia mutation site ( figure 1 ). From the current structure of I-SceI, it can be found that some amino acids in I-SceI are in direct contact with the DNA sequence. By gradually changing the sequence of bases, and then screening these directly contacted amino acids, and finding the most specific new combination amino acid binding site (see figure 2 ).

[0040] During the recognition process of I-SceI and DNA sequence, ATAAC in the DNA sequence is an important part of protein cleavage. When these bases are changed, the cleavage efficiency will be significantly reduced, which can be screened by...

Embodiment 2

[0055] Embodiment 2, functional verification of chimeric nuclease TALE-I-SceI

[0056] 1. Improving the efficiency of gene targeting in the human genome

[0057] The modified fusion protein has the effect of cutting genomic DNA. In the absence of a donor, the DSB will be repaired by the NEHJ repair method in vivo, and indels or mutations will be generated at the cutting site.

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Abstract

The invention discloses a fusion protein of chimeric nuclease for specific recognition and repair of a beta thalassemia beta-globin gene, wherein the fusion protein is a protein obtained by replacing an FokI part of a TALE protein with a transformed I-SceI protein; the TALE protein has an amino acid sequence shown in a sequence 4 in a sequence table; the transformed I-SceI protein has an amino acid sequence being 706-740th amino acids from an N' tail end of a sequence 2 in the sequence table. Experiments show that the advantages of the TALE-ISceI fusion enzyme in clinical application and show the feasibility of application of the TALE-ISceI fusion enzyme in radical treatment of beta-thalassemia.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a chimeric nuclease that specifically recognizes and repairs the beta-globin gene of beta thalassemia. Background technique [0002] Thalassemia is a genetic disease with high incidence and wide distribution in China and the world. Normal hematopoietic stem cells have the ability to differentiate into blood cells. In patients with thalassemia, their red blood cells are abnormal, fragile and easy to die, and their oxygen-carrying capacity is also insufficient. Therefore, hematopoietic stem cell transplantation (Hematopoieticstemcelltransplantation, HSCT) is currently the most effective cure for β-thalassemia major The methods include bone marrow hematopoietic stem cell transplantation (BMT), peripheral blood stem cell transplantation (PBSCT) and cord blood stem cell transplantation (CBSCT). However, lack of donors and rejection have severely hindered the w...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/22C12N15/62C12N15/85A61K38/46A61K48/00A61P7/06
CPCA61K38/46A61K48/00C07K14/00C07K19/00C12N9/22C12N15/62C12N15/85
Inventor 纪家葵林建飞
Owner TSINGHUA UNIV
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