Single base mutation method and adopted system

Abstract

The invention relates to a single base mutation method and an adopted system. The single base mutation method comprises the following steps that sgRNA is designed for a point mutation site of a gene to be mutated and CRISPR / Cpf1 plasmid is constructed; a base sequence near a mutation point to be mutated into a gene is intercepted, and nonsense mutation is introduced into the base sequence to synthesize donor1; the CRISPR / Cpf1 plasmid and donor1 are co-transfected to a cell containing the gene to be mutated so as to obtain a cell containing nonsense mutation; Re-sgRNA is designed for a point mutation site of a sequence containing nonsense mutation in the cell containing nonsense mutation, and Re-CRISPR / Cpf1 plasmid is constructed; the base sequence near the mutation point to be mutated into the gene is intercepted to synthesize donor2; and the Re-CRISPR / Cpf1 plasmid and donor2 are co-transfected to the cell containing nonsense mutation to obtain the cell containing genes to be mutated into. According to the single base mutation method, the purpose of traceless repair can be achieved.

Description

[0001] This case is a divisional application with an application date of 2019 / 3 / 26, an application number of 2019102324760, and a patent name of a single base mutation method and the system used. technical field [0002] The invention belongs to the field of CRISPR / Cpf1 gene therapy, and specifically relates to a single base mutation method and a system adopted. Background technique [0003] β-thalassemia (β-thalassemia) is a disease caused by a decrease in or failure to synthesize the globin peptide chains that make up hemoglobin HbA (a2β2) due to mutations or deletions in the β-globin gene, resulting in an imbalance in the synthesis of a and β peptide chains. An autosomal recessive genetic disorder. β-thalassemia gene is often the most common point mutation, β-globin non-coding region IVS-II-654 point mutation (C → T) is one of the earliest reported point mutations associated with β-thalassemia disease, the pathogenicity Mutations can affect processes such as mRNA splicin...

AI Technical Summary

Problems solved by technology

[0007] In 2015, the patent applicant used CRISPR gene editing technology for the first time in the world to edit and repair the HBB gene of abnormally fertilized human 3PN fertilized eggs. It was observed that the cutting efficiency of CRISPR on the target gene in human embryos reached 51.9%, but at the same time, it was found that off-target was obvious

However, the gene repair efficiency is only 23%, and the embryos obtained after repair are still chimeras, and the repaired blastomeres are only 20%, so it is difficult to achieve complete cure, and the operation is complicated and cumbersome

In addition, this method also has a high off-target rate

The above characteristics will greatly limit the use of this technology in the gene editing treatment of β-thalassemia

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