Intron abnormal splicing repair method

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...

AI Technical Summary

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 cells, which will reduce the curative effect, that is, it may not be possible to achieve the purpose of lifelong cure

In addition, the high-concentration and high-quality lentivirus required in clinical practice has extremely high requirements on equipment and technology, so it is difficult to reduce the cost

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