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gRNA, gene editing system, expression vector and gene editing kit for repairing HBB1 gene point mutation

A gene editing and expression vector technology, applied in the field of gRNA, can solve the problems of low induction rate of iPS cells, high R&D cost, limited clinical application prospects, etc., to avoid matching limitations and immune rejection, and reduce treatment costs.

Inactive Publication Date: 2019-03-19
广东赤萌医疗科技有限公司
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  • 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

Method used

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  • gRNA, gene editing system, expression vector and gene editing kit for repairing HBB1 gene point mutation
  • gRNA, gene editing system, expression vector and gene editing kit for repairing HBB1 gene point mutation
  • gRNA, gene editing system, expression vector and gene editing kit for repairing HBB1 gene point mutation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1: Cutting the HBB1 gene using the CRISPR-Sp Cas9 gene editing system

[0065] 1.1 gRNA preparation

[0066] (1) Design the target sequence of the gRNA of 20nt according to the HBB1 gene sequence;

[0067] (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) ;

[0068] (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.

[0069] The positive sense strand of the target sequence of th...

Embodiment 2

[0095] Example 2: Using the CRISPR-Sa Cas9 gene editing system to cut the HBB1 gene

[0096] 2.1 gRNA preparation

[0097] (1) Design the target sequence of the gRNA of 21nt according to the HBB1 gene sequence;

[0098] (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);

[0099] (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.

[0100] The sense strand of the target sequence of the gRNA designed for the HBB1 gene in Tabl...

Embodiment 3

[0125] Example 3 Homologous recombination vector construction and site-directed homologous recombination of HBBI gene

[0126] 3.1 Homologous recombination vector construction

[0127] 3.1.1 Construction of the upstream homology arm vector of SEQ ID NO:17

[0128] (1) Using the HEK293T cell genome as a template, using SEQ ID NO: 29 and SEQ ID NO: 30 as primers to amplify the upstream homology arm of SEQ ID NO: 17, and the PCR product was detected by 1% agarose gel electrophoresis and then recovered by cutting the gel. Purpose Fragment;

[0129] Table 3 HBB1 gene target site SEQ ID NO:17 upstream and downstream homology arms and tNGFR amplification primers

[0130]

[0131] (2) Digest plasmid pDonor-TALEN-EGFP with SnaBI and SalI-HF, digest at 37°C for 1 hour, purify and recover by 1% agarose gel electrophoresis after digestion;

[0132] (3) Homologous recombination was carried out between the above-mentioned digested product and the upstream homology arm PCR product of S...

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Abstract

The invention provides gRNA, a gene editing system, an expression vector and a gene editing kit for repairing HBB1 gene point mutation. Two common mutation sites of HBB genes can be repaired through one-time gene editing aiming at fixed-point repair of beta 17 (A ->T) and beta 41-42 (-TCTT), and thus treatment methods of thalassemia and sickle-shaped anemia are expanded; and in addition, the editing is directly preformed in hematopoietic stem cells (HSC), and compared with gene editing of iPS cells in the prior art, the safety risks of cell carcinogenesis and mutation are avoided, and the 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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IPC IPC(8): C12N15/113C12N15/90C12N15/85C12N5/10A61K31/7105A61K48/00A61P7/06
CPCA61K31/7105A61P7/06C12N5/0647C12N15/113C12N15/85C12N15/907C12N2310/10C12N2510/00C12N2310/20
Inventor 祝海宝刘洁黄雨亭陶米林梁福才
Owner 广东赤萌医疗科技有限公司
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