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gRNA for knocking out BCL11A genes or BCL11A gene enhancers, gRNA composition and electrorotation method

A composition and gene technology, applied in the field of gRNA and gRNA composition, can solve the problems of cytotoxicity, difficulty in achieving effective knockout, low efficiency of electroporation and editing, and achieve convenient use, low synthesis cost, simple design and use Effect

Pending Publication Date: 2019-05-03
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

The knockout efficiency of the screened gRNA is only about 10% by electrotransferring a single carrier into HSC cells by using the single-transfer technology. purpose of removal
Other sequences carried in the vector can cause certain toxicity to cells

Method used

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  • gRNA for knocking out BCL11A genes or BCL11A gene enhancers, gRNA composition and electrorotation method
  • gRNA for knocking out BCL11A genes or BCL11A gene enhancers, gRNA composition and electrorotation method
  • gRNA for knocking out BCL11A genes or BCL11A gene enhancers, gRNA composition and electrorotation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Using CRISPR-Sp Cas9 technology to mutate BCL11A gene or BCL11A gene enhancer

[0048] 1.1 gRNA preparation

[0049] (1) Design a 20nt gRNA sequence according to the sequence of the BCL11A gene and the BCL11A gene enhancer, the target sequence of the gRNA is as in SEQ ID NO:1-SEQ ID NO:18, SEQ ID NO:31-SEQ ID NO:34 as shown in one of

[0050] (2) Synthesize the sense strand and antisense strand of the DNA sequence corresponding to the 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) ;

[0051] (3) Mix the sense strand and antisense strand of the DNA sequence corresponding to the above gRNA, treat at 90°C, cool naturally to room tem...

Embodiment 2

[0083] Example 2 Using CRISPR-Sa Cas9 technology to mutate the BCL11A gene enhancer

[0084] 2.1 gRNA preparation

[0085] (1) Design a 20nt gRNA sequence according to the sequence of the BCL11A gene enhancer, the target sequence of the gRNA is as shown in one of SEQ ID NO:24-SEQ ID NO:27;

[0086] (2) Synthesize the sense strand and antisense strand of the DNA sequence corresponding to the 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) ;

[0087] (3) Mix the sense strand and antisense strand of the DNA sequence corresponding to the above gRNA, treat at 90°C, cool naturally to room temperature for annealing, and synthesize double-stranded DNA with...

Embodiment 3

[0117] Example 3 Sequencing Analysis of BCL11A Gene Mutation Efficiency and Mutation Type

[0118] Select the gRNA with higher mutation efficiency in Example 1 or Example 2, and further analyze the mutation efficiency and mutation type. Taking SEQ ID NO:16, SEQ ID NO:31, SEQ ID NO:32 and SEQ ID NO:34 in Example 1; SEQ ID NO:25 and SEQ ID NO:27 in Example 2 are examples to carry out the following experimental operations .

[0119] 3.1 Measure the concentration of the purified PCR product obtained in 1.5(4) in Example 1 or 2.5(4) in Example 2 for subsequent use;

[0120] 3.2 Choose Kangwei Century Master PCR Mix to add an adenine (A) to the 3'-end of the PCR product: take 2 μg of the PCR product in 3.1 and mix it with the Master PCR Mix at a ratio of 1:1 (V:V), and react at 72°C for 30 minutes ;

[0121] 3.3 Use 1% agarose gel to electrophoresis the reaction product in 3.2, cut the gel to recover the target fragment and measure the concentration of the recovered product;

[...

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Abstract

The invention provides gRNA for knocking out BCL11A genes or BCL11A gene enhancers, a gRNA composition, an expression vector, CRISPR-Cas9 RNP, a CRISPR-Cas9 RNP composition, a CRISPR-Cas9 system, an electrorotation method, a reagent kit and applications thereof. A CRISPR-Cas9 technique is adopted to perform targeted cutting on hemopoietic stem cells, so that the BCL11A gene expression amount is reduced, and the hemochrome expression quantity of fetuses is high. The technique is hopeful to become a new means for treating thalassaemia and sickle cell anemia. The CRISPR-Cas9 technique is adoptedto realize mutation of BCL11A genes. The design is simple, the use is convenient, the cost is low, and the efficiency is high.

Description

technical field [0001] The present invention relates to a gRNA, gRNA composition and electroporation method for knocking out BCL11A gene or BCL11A gene enhancer. Background technique [0002] Hemoglobinopathies are genetic diseases that affect the health and even the survival rate of newborns. There are more than 330,000 newborns every year. There are mainly two types of sickle cell anemia and thalassemia. Thalassemia is a hereditary chronic anemia caused by a globin gene defect. The β and α types are more common. At least 81 kinds of α-thalassemia gene mutations (46 point mutations, 35 deletion mutations) have been found; β-thalassemia gene mutations have at least 186 kinds, mainly point mutations. my country is one of the countries with a high incidence of thalassemia. Cases are distributed in Guangdong, Guangxi, Hainan, Guizhou, Yunnan, Sichuan, Chongqing, Fujian, Hunan, Hubei, Jiangxi and other places. Among them, the thalassemia gene defect rate in Guangdong and Guangx...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/90C12N15/85C12N15/12C12N5/10A61K48/00A61K35/28A61P7/06
Inventor 祝海宝陶米林黄雨亭张栩琳阮锦辉
Owner 广东赤萌医疗科技有限公司
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