CRISPR single-base restoration system and application thereof

Abstract

The invention discloses a CRISPR single-base restoration system. The CRISPR single-base restoration system is capable of expressing eukaryotic expression vectors of sgRNA and Cas9 and single strandedoligonucleotide as a donor template, wherein the base sequence of single stranded oligonucleotide is basically the same as the base sequence between bases 30-60 on two sides of a to-be-restored singlebase, and the differences between the two base sequences are that the base corresponding to the to-be-restored single base is a non-mutation wild base, and nonsense mutation occurs on one side of theto-be-restored single base. The invention further discloses application of the CRISPR single-base restoration system in the preparation of drugs for treating beta thalassemia caused due to CD17A-T point mutation of a beta bead protein gene. According to the CRISPR single-base restoration system, a CRISPR / Cas expression plasmid and a donor are commonly transfected into cells, the traceless directional transformation of gene mutation sites in beta17 thalassemia is specifically induced in a 293TT cell, and the method has very important clinical application values in the execution of gene editingaccurate treatment on diseases based on single-gene point mutation.

Description

technical field [0001] The invention relates to the technical field of gene editing, in particular to a CRISPR single-base repair system and its application in the treatment of β17 thalassemia genetic diseases caused by single gene point mutations. 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. The point mutation of β-thalassemia gene is often the most common, and the CD17(A→T) point mutation of β-globin gene is one of the earliest reported point mutations related to β-thalassemia disease. [0003] Thalassemia is currently treated with lifelong blood transfusions, yet the vast majority of children die before adulthood despite aggressive treatment. Another treatment option is bone...

AI Technical Summary

Problems solved by technology

[0005] In 2015, it was reported that for the first time in the world, CRISPR / Cas9 gene editing technology was used to repair abnormally fertilized human 3PN fertilized eggs by HBB gene editing. Obvious miss

Partial sulfur modification of terminal nucleotides can greatly improve the ability of ODN to resist nuclease degradation without having to carry out sulfur modification on the entire ODN. Hybridization forms a stable double-strand structure, but studies have shown that comparing the changes in its Tm value, it is found that the stability and melting temperature (Tm) of this double-strand has a certain decrease compared with the double-strand of the corresponding normal oligonucleotide. In addition, thiooligonucleotides are often mildly toxic to normal cells

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