GRNA, expression vector, knockout system and kit for knocking out CXCR4 gene

A technology of expression vectors and kits, applied in the field of genetic engineering, can solve problems such as high cost of use, cumbersome and complicated design and use, and achieve the effects of improving feasibility, reducing safety risks, and reducing off-target efficiency

Inactive Publication Date: 2018-04-10
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

To use ZFN to knock out CXCR4, it is first necessary to design a 27nt gene-specific modification site, and also to design the amino acid sequence of the zinc finger nuclease that can recognize the CXCR4 gene, and insert these sequences into the corresponding zinc finger nuclease coding basket Among them, the whole design and use are cumbersome and complicated, and the cost of use is high

Method used

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  • GRNA, expression vector, knockout system and kit for knocking out CXCR4 gene
  • GRNA, expression vector, knockout system and kit for knocking out CXCR4 gene
  • GRNA, expression vector, knockout system and kit for knocking out CXCR4 gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 gRNA preliminary screening

[0035] 1. gRNA preparation

[0036] (1) designing a 20nt gRNA sequence according to the sequence of the CXCR4 gene, the target sequence of the gRNA is shown in one of SEQ ID NO:1-SEQ ID NO:24;

[0037] (2) Synthesize the sense strand and 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, then add C at the 3'-end of the antisense strand);

[0038] (3) The above-mentioned gRNA sense strand and antisense strand were mixed, treated at 90°C, cooled naturally to room temperature for annealing treatment, and double-stranded gRNA with sticky ends was synthesized.

[0039] 2. Carrier preparation

[0040] (1)pX458( figu...

Embodiment 2

[0066] Embodiment 2 gRNA combinatorial screening

[0067] 1. Preparation of gRNA combination

[0068] (1) Select the gRNA with higher mutation efficiency analyzed by T7E1 in Example 1, and select a paired gRNA within 200 bp of the DNA complementary strand of the target sequence it binds to, and the paired combination is shown in Table 2;

[0069] Table 2 gRNA pairing combination

[0070]

[0071]

[0072] (2) From the gRNA sense strand and antisense strand synthesized in 1 (2) of Example 1, select the fragments required for the above pairing, and anneal according to the method of 1 (3) of Example 1 to synthesize the double gRNA with cohesive ends. stranded gRNA.

[0073] 2. Carrier preparation

[0074] (1) pX461 plasmid (such as Figure 4 ) amplify and extract, and measure the plasmid concentration;

[0075] (2) Digest pX461 with restriction endonuclease Bbs I, digest at 37°C for 1 hour, and then add loading buffer to terminate the reaction.

[0076] (3) The linear...

Embodiment 3

[0097] Embodiment 3 and the contrast of prior art

[0098] 1. Preparation of gRNA

[0099] (1) The sense strand and antisense strand of the target sequence corresponding to the gRNA with the highest mutation efficiency in the outsourced synthesis comparison file (add cacc to the 5'-end of the sense strand, if the first nucleotide at the 5'-end of the sense strand is not Guanine G, add caccG at the 5'-end of the sense strand; 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, then add aaac at the 5'-end of the antisense strand The 3'-end of the chain adds C), and the DNA sequence of the sense chain is shown in Table 2. In Example 1, gRNAs with higher mutation efficiency analyzed by T7E1 (the corresponding sense strands of the target sequences are SEQ ID NO:7, SEQ ID NO:20, and SEQ ID NO:24) were selected for comparison.

[0100] Table 2 The positive-sense strand of the target sequence corresponding to th...

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Abstract

The invention discloses a gRNA, a knockout system and a kit for knocking out CXCR4 gene. A target site with high cutting efficiency is screened, the gRNA specifically targeting a target gene is designed and prepared, the CXCR4 gene can be effectively knocked out by CRISPR-Cas9 technology, HIV virus co-receptor CXCR4 on cell surface can be knocked out by CRISPR-nickase gene editing technology, theoff-target efficiency can be greatly reduced under the premise of effective CXCR4 gene knockout, the feasibility of clinical use is improved, and security risks can be reduced.

Description

technical field [0001] The invention relates to a gRNA, an expression vector, a gene editing system, a kit for knocking out the CXCR4 gene and their related uses, belonging to the field of genetic engineering. Background technique [0002] Acquired immunodeficiency syndrome (AIDS) is an immune system disease caused by human immunodeficiency virus (Human Immunodeficiency Virus, HIV) infection, and is a very harmful global infectious disease. HIV takes the most important T lymphocytes in the human immune system as the main attack target, resulting in the loss of immune function, making it susceptible to various diseases, and the mortality rate is as high as 99%-100%. [0003] The incubation period of HIV in the human body can be as long as 8-10 years. Many HIV-infected people can live without symptoms before developing into AIDS patients, but the virus always exists in the human body and cannot be cured. Although many medical researchers around the world are focusing on the p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/90C12N15/85A61K48/00A61P31/18
CPCA61K48/005C07K14/7158C12N9/22C12N15/113C12N15/85C12N15/907C12N2310/10C12N2800/107C12N2810/10
Inventor 梁福才祝海宝罗思施黄雨亭陶米林刘方方唐忆琳
Owner 广东赤萌医疗科技有限公司
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