SgRNA and use thereof in repairing abnormal splicing of introns

An intronic and abnormal technology, applied in the field of genetic engineering, can solve the problems of inability to achieve lifelong cure, extremely high requirements for equipment and technology, and decline in curative effect

Pending Publication Date: 2021-02-23
EAST CHINA NORMAL UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Among them, the use of lentiviral vectors for gene therapy has shown great potential, but the semi-random vector integration method has cancer risk
At the same time, the expression elements in the lentivirus will be gradually silenced during the long-term homing and self-renewal process of hematopoietic stem

Method used

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  • SgRNA and use thereof in repairing abnormal splicing of introns
  • SgRNA and use thereof in repairing abnormal splicing of introns
  • SgRNA and use thereof in repairing abnormal splicing of introns

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Embodiment Construction

[0019] On the one hand, the present invention provides a method for repairing the abnormal splicing of introns caused by the mutation of HBB (beta-globin gene) IVS2-654 C>T in cells, and the IVS2-654 C>T can cause additional The splice donor site of said method comprises the step of using CRISPR-Cas9 system to carry out gene editing to HBB to delete said additional splice donor site;

[0020] The CRISPR-Cas9 system includes Cas9 and sgRNA targeting target sequence;

[0021] The sgRNA includes sgRNA-U and sgRNA-D,

[0022] The targeting site of the sgRNA-U is within the range of the starting site of the second intron of the HBB gene from the upstream 21 bp of the IVS2-654 C>T site to the IVS2-654 C>T site,

[0023] The target site of the sgRNA-D is within the range from the 71 bp downstream of the IVS2-654 C>T site to the second intron termination site of the HBB gene where the IVS2-654 C>T site is located.

[0024] Further, the targeting sequence of the sgRNA-U is any one or...

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Abstract

The invention discloses a sgRNA and use thereof in repairing abnormal splicing of introns. The sgRNA is used for repairing abnormal splicing of introns caused by a HBB (a beta-globin gene) IVS2-654 C>T mutation and comprises a sgRNA-U and a sgRNA-D. A targeting site of the sgRNA-U is located in a range from an upstream 21 bp of the IVS2-654 C>T site to a starting site of a second intron of the HBBgene where the IVS2-654 C>T site is located. A targeting site of the sgRNA-D is located in a range from a downstream 71bp of the IVS2-654 C>T site to a termination site of the second intron of the HBB gene where the IVS2-654 C>T site is located. An existing gene editing technology can be used for repairing a blood transfusion-dependent beta-thalassemia IVS2-654 C>T, has high editing efficiency, and can efficiently modify a persistent and balanced hematopoietic system for autologous hematopoietic stem cells.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a sgRNA and its application in repairing abnormal splicing of introns. Background technique [0002] β-thalassemia is a common genetic disease caused by abnormal hemoglobin in adults due to β-globin gene defects. There are about 30 million carriers of the "thalassemia" gene in my country, involving nearly 30 million families and 100 million people. Among them, severe and about 300,000 patients with intermediate "thalassemia". Among them, the IVS2-654 C>T genotype is a relatively common type of "thalassemia" in my country. The pathogenesis is that the 654th base in the second intron of the HBB gene has a C>T mutation, resulting in abnormalities. splice site, resulting in an extra 73nt exon in β-globin mRNA and premature termination of translation. [0003] At present, patients with intermediate and severe thalassemia need long-term blood transfusion and iron remo...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/90C12N15/12C12N15/113C12N5/10
CPCC12N15/85C12N15/907C07K14/805C12N15/113C12N5/0647C12N2800/107C12N2310/20C12N2320/33C12N2510/00
Inventor 吴宇轩杨菲刘明耀张亮
Owner EAST CHINA NORMAL UNIVERSITY
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