Intron abnormal splicing repair method

A repair method and intron technology, applied in the field of genetic engineering, can solve problems such as inability to achieve lifelong cure, extremely high requirements for equipment and technology, and semi-random

Pending Publication Date: 2020-12-22
EAST CHINA NORMAL UNIVERSITY +1
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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

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  • Intron abnormal splicing repair method
  • Intron abnormal splicing repair method
  • Intron abnormal splicing repair method

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Embodiment Construction

[0021]In one aspect, the present invention provides a method for repairing abnormal splicing of introns caused by the mutation of HBB (beta-globin gene) IVS2-654C>T in cells, and the IVS2-654C>T can cause additional splicing A donor site, the method comprising the step of gene editing HBB using the CRISPR-Cas9 system, the gene editing of HBB using the CRISPR-Cas9 system to inactivate the additional splicing donor site, the CRISPR-Cas9 The system includes Cas9 and at least one sgRNA targeting the target sequence.

[0022] The inactivation includes destroying the function of the above-mentioned additional splice donor site through base insertion, deletion, alteration, frameshift mutation or knockout.

[0023] Further, the targeting sequence of the sgRNA is 20 bp upstream of the IVS2-654C>T site to 70 bp downstream of the IVS2-654C>T site.

[0024] In one embodiment, the sgRNA includes sgRNA-1; in other embodiments, the sgRNA includes sgRNA-1 and sgRNA-2.

[0025] The targeting...

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Abstract

The invention discloses an intron abnormal splicing repair method. The method is a technology for targeted knockout of an abnormal mutation site IVS2-654 C> T in beta-thalassemia (thalassemia) by using a CRISPR-Cas9 gene editing technology, and comprises the following steps: designing and synthesizing a guide RNA sequence (sgRNA) capable of identifying and guiding a Cas9 protein to a target sequence of a target gene; And electrically transferring and introducing an sgRNA and Cas9 protein mixture into beta-thalassemia IVS2-654 C>T hematopoietic stem cells to efficiently destroy an abnormal splicing mutation site, so that normal shearing and expression of a beta-globin gene are recovered. The blood transfusion dependent beta-thalassemia IVS2-654 C>T can be edited by utilizing the existing gene editing technology, the editing efficiency is high, and the edited hematopoietic stem cells of the patient can reconstruct a blood system of the patient and treat thalassemia diseases after autotransplantation.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a method for repairing abnormal intron splicing sites. Background technique [0002] In recent years, an acquired immune mechanism used to resist the invasion of foreign DNA fragments such as phages and plasmids in bacteria and archaea has been elucidated. The system consists of Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (CAS) genes. The immune interference process of the CRISPR system mainly includes three stages: adaptation, expression, and interference. In the adaptation phase, the CRISPR system will integrate a short DNA fragment from a phage or plasmid between the leader sequence and the first repeat sequence, and each integration is accompanied by the duplication of the repeat sequence, thereby forming a new repeat-spacer unit. In the expression phase, the CRISPR locus is transcribed into a CRISPR RNA (crRNA) precu...

Claims

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Application Information

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IPC IPC(8): C12N15/90C12N9/22C12N15/113
CPCC12N15/907C12N9/22C12N15/113C07K14/805C12N2310/20
Inventor 吴宇轩杨菲李大力刘明耀
Owner EAST CHINA NORMAL UNIVERSITY
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