Method of performing gene correction on hemopoietic stem cells of patient with Mediterranean anemia

Abstract

The invention discloses a method of performing gene correction on hemopoietic stem cells of a patient with Mediterranean anemia. The method is characterized by comprising the steps of I, selecting CRISPR target near an HBB gene-28 (A / G) site, and designing a gRNA sequence; II, constructing a template plasmid, and inserting a dual-ITR (inverted terminal repeat) sequence of piggyBac between upstreamand downstream 500bp sequences of the HBB gene CRISPR target; III, co-transfecting the hemopoietic stem cells of the patient with Mediterranean anemia through a transfection reagent, the template plasmid, gRNA and Cas9 carrier, and performing puromycin screening on a cell medium; IV, authenticating and verifying a resistant gene. The CRISPR / Cas9 system and piggyBac are jointly utilized; the hemopoietic stem cells of the patient with Mediterranean anemia can be transfected effectively; homologous recombination efficiency can be improved; -28(A / G) mutation site of the patient with Mediterraneananemia can be corrected effectively.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a method for gene modification of hematopoietic stem cells of thalassemia patients. Background technique [0002] Beta thalassemia is an autosomal recessive genetic disorder. The cause of the disease is the mutation of the HBB (hemoglobin beta) gene, resulting in incomplete function or even non-expression of β-globin. The disease is mainly distributed in the Mediterranean region (from the Middle East to Southeast Asia), and is the most common and most harmful blood genetic disease in the southern provinces of my country. -28(A / G) is one of the common mutation sites in Chinese thalassemia population. Both HBBs on the β chain of patients with severe thalassemia have severe -28(A / G) mutations. Begins to show severe anemia, stunted growth and development, often requiring regular blood transfusions and iron excretion to sustain life. [0003] Currently, allogeneic hematopoietic s...

AI Technical Summary

Problems solved by technology

However, in the treatment of other genetic diseases, it has been found that gene editing of the host genome with viral vectors may have off-target effects, causing damage to other genes, and there are potential risks; the market needs a method that can improve the efficiency of homologous recombination The method of gene correction, the present invention solves such problems

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