sgRNA editing HBB-28 mutation site based on CRISPR/Cas9 technology, vector and application thereof

Abstract

An sgRNA editing HBB-28 mutation site based on CRISPR / Cas9 technology is disclosed, the nucleotide sequence of which is shown in SEQ ID NO.1 or SEQ ID NO.2. The invention also provides a vector associated therewith, a cell, a gene editing kit and an application thereof, in particular a method for editing the HBB-28 mutation site based on the CRISPR / Cas9 technology, comprising constructing a CRISPR / Cas9 expression vector use sgRNA having a nucleotide sequence represented by SEQ ID NO.1 or SEQ ID NO.2. The invention can specifically induce HBB-28 Traceless modification of gene mutation sites inthalassemia can selectively introduce monoallelic and biallelic sequence changes with high efficiency and precision, which greatly promotes the precise repair of thalassemia.

Description

technical field [0001] The invention belongs to the field of biomedicine, and specifically relates to a method for constructing a CRISPR plasmid targeting pathogenic point mutations of HBB-28 thalassemia, and a scheme for efficiently and accurately repairing a single-base mutation donor. 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 (α2β2) due to mutations or deletions in the β-globin gene, resulting in an imbalance in the synthesis of α- and β-peptide chains. An autosomal recessive genetic disorder. β-thalassemia gene is often the most common point mutation, β-globin transcriptional revelation region TATA box-28 point mutation (A→G) is one of the earliest reported point mutations associated with β-thalassemia disease. [0003] According to reports, there are about 400 million thalassemia gene carriers worldwide. In my country, the disease occur...

AI Technical Summary

Problems solved by technology

[0005] In 2015, for the first time in the world, CRISPR / Cas9 gene editing technology was used to repair the HBB gene of abnormally fertilized human 3PN fertilized eggs. It was observed that the efficiency of CRISPR / Cas9 cutting the target gene in human embryos reached 51.9%, but at the same time, it was found that off-target was obvious

The gene repair efficiency is only 23%, and the embryos obtained after repair are still chimeras, and the repaired blastomeres are only 20%, 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

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