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GRNA and gene editing system for repairing HBB1 gene site mutation, expression vector and gene editing kit

A technology of gene editing and expression vectors, applied in the field of gRNA, can solve the problems of high safety risk of non-target gene off-target mutations, high R&D costs, and limited clinical application prospects, so as to avoid matching limitations and immune rejection, and reduce treatment costs. cost effect

Pending Publication Date: 2019-03-29
广东赤萌医疗科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

According to the idea of ​​the existing technology, it is necessary to design and develop a gene editing system for each mutation type, the workload is cumbersome, and the research and development cost is high
[0011] 2. The existing technology uses iPS cells for gene editing, the induction rate of iPS cells is low, and there are safety risks such as carcinogenesis and gene mutation, which limit its clinical application prospects
[0012] 3. The existing technology uses the CRISPR-Cas9 system to introduce double-strand breaks in the target gene to mediate homologous recombination, which has a high safety risk of causing off-target mutations in non-target genes

Method used

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  • GRNA and gene editing system for repairing HBB1 gene site mutation, expression vector and gene editing kit
  • GRNA and gene editing system for repairing HBB1 gene site mutation, expression vector and gene editing kit
  • GRNA and gene editing system for repairing HBB1 gene site mutation, expression vector and gene editing kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1: Using the CRISPR-Sp Nickase gene editing system to cut the HBB1 gene

[0069] 1.1 gRNA preparation

[0070](1) Design the target sequence of the gRNA of 20nt according to the HBB1 gene sequence;

[0071] (2) Synthesize the sense strand and antisense strand of the target sequence of gRNA respectively (add cacc at the 5'-end of the sense strand, if the first nucleotide at the 5'-end of the sense strand is not guanine G, then add cacc at the 5'-end of the sense strand Add caccG at the 5'-end; add aaac at the 5'-end of the antisense strand, if the first nucleotide at the 5'-end of the sense strand is not guanine G, add C at the 3'-end of the antisense strand) ;

[0072] (3) The sense strand and antisense strand of the above gRNA were mixed, treated at 90°C, cooled naturally to room temperature for annealing treatment, and the target sequence of double-stranded gRNA with sticky ends was synthesized.

[0073] The positive sense strand of the target sequence of t...

Embodiment 2

[0098] Example 2: Using the CRISPR-Sa Nickase gene editing system to cut the HBB1 gene

[0099] 2.1 gRNA preparation

[0100] (1) Design the target sequence of the gRNA of 21nt according to the HBB1 gene sequence;

[0101] (2) Synthesize the sense strand and the antisense strand of the gRNA target sequence respectively (add cacc at the 5'-end of the sense strand, if the first nucleotide at the 5'-end of the sense strand is not guanine G, then add cacc at the 5'-end of the sense strand Add caccG at the '-end; add aaac at the 5'-end of the antisense strand, if the first nucleotide at the 5'-end of the sense strand is not guanine G, add C at the 3'-end of the antisense strand);

[0102] (3) Mix the sense and antisense strands of the above-mentioned gRNA target sequence, treat at 90°C, cool naturally to room temperature for annealing, and synthesize double-stranded gRNA with sticky ends.

[0103] The sense strand of the target sequence of the gRNA designed for the HBB1 gene in T...

Embodiment 3

[0129] Example 3 Using the CRISPR-Sp Nickase gene editing system to cut the HBB1 gene

[0130]The gRNA with higher mutation efficiency screened out in Example 1 was used to construct the Sp Nickase vector. The following examples take gRNA target sequences SEQIDNO: 3 and SEQIDNO: 9 as examples to demonstrate the implementation effect of other gRNAs in the same way.

[0131] 3.1 Carrier preparation

[0132] (1) pX461 plasmid ( Figure 8 ) amplify and extract, and measure the plasmid concentration;

[0133] (2) According to 1.2(2)-(3), pX461 was digested with restriction endonuclease Bbs I, recovered from the gel, and the concentration of the recovered product was measured, and stored at -20°C for future use.

[0134] 3.2 Connection conversion

[0135] (1) Ligate the linearized pX461 vector recovered by cutting the gel with the gRNA double strands of SEQ ID NO:3 and SEQ ID NO:9 synthesized in 1.1;

[0136] (2) According to 1.3(2)-(6), transform the ligation product into Esche...

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Abstract

The invention provides a gRNA and gene editing system for repairing HBB1 gene site mutation, an expression vector and a gene editing kit. The gRNA aims at site-directed repair of [beta]17 (A->T) and [beta]41-42 (-TCTT), two common mutation sites of an HBB gene are repaired through one-time gene editing, and treatment methods of thalassemia and sickle anemia are expanded; in addition, the gene editing system is used for editing directly in hematopoietic stem cells (HSC); compared with gene editing on iPS cells in the prior art, the safe risks of cell carcinogenesis and mutation are avoided, andthe clinical use is safer.

Description

technical field [0001] The invention relates to a gRNA, a gene editing system, an expression vector and a gene editing kit for repairing a point mutation of the HBB1 gene. Background technique [0002] my country is a country with a high incidence of thalassemia, and the cases are mainly distributed in Guangdong, Guangxi, Hainan, Guizhou, Yunnan, Sichuan, Chongqing, Fujian, Hunan, Hubei, Jiangxi and other places. Among them, the south has a high incidence of thalassemia, while Guangdong and Guangxi provinces have thalassemia gene defect rates as high as 10% and 20% respectively, accounting for more than 2 / 5 of the total number of cases in the country, which has become a social public health problem. And because many provinces have not included thalassemia screening in the routine items of pregnancy checkup, and the mandatory premarital checkup system has been canceled in recent years, and the level of awareness of the disease among grassroots medical staff in some areas is n...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/85C12N5/10A61K35/28A61P7/06
CPCA61K35/28A61P7/06C12N5/0647C12N15/113C12N15/85C12N2310/10C12N2510/00
Inventor 祝海宝黄雨亭刘洁梁福才陶米林
Owner 广东赤萌医疗科技有限公司
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