Single base mutation method and adopted system

Abstract

The invention relates to a single base mutation method and an adopted system. For a point mutation site of a to-be-mutated gene, an sgRNA is designed, and a CRISPR / Cpf1 plasmid is constructed; a basesequence near the mutation site of the to-be-mutated gene is intercepted, and a nonsense mutation is introduced into the base sequence to synthesize donor1; a cell containing the to-be-mutated gene isco-transfected with the CRISPR / Cpf1 plasmid and the donor1 to obtain a cell containing the nonsense mutation; for the point mutation site of the sequence containing the nonsense mutation in the cellcontaining the nonsense mutation, Re-sgRNA is designed, and an Re-CRISPR / Cpf1 plasmid is constructed; the base sequence near the mutation site of the to-be-mutated gene is intercepted to synthesize donor2; the cell containing the nonsense mutation is co-transfected with the Re-CRISPR / Cpf1 plasmid and the donor2 to obtain the cell containing the to-be-mutated gene. The single base mutation method and the adopted system can achieve the purpose of non-trace repair.

Description

technical field [0001] 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 [0002] 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 splicing that cannot be carried out accurately to form abnormal mRNAs, resulting in beta thalassemia. [0003] According to reports, there are about 400 million thalassemia gene carriers worldwide. In my...

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Problems solved by technology

[0006] 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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