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Method for knocking out microRNA gene family by utilizing CRISPR-Cas9 specificity

A gene family, pgem-cas9 technology, applied in the field of specific targeting sgRNA design, can solve the problems of repeated injections, waste of manpower and material resources, restriction of embryos, etc., to achieve simple operation technology, reduced capital investment, and reduced unreliability Effect

Inactive Publication Date: 2015-05-27
HANGZHOU NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most microRNAs regulate gene expression in the form of microRNA families; knocking out a microRNA alone does not completely change the function of the target gene
However, using the traditional method to construct a microRNA family knockout animal model is complicated and labor-intensive; the use of artificially synthesized microRNA antagonists to transfect cells or intravenously inject them into animals is obviously insufficient: the cost is high and repeated injections are required, among which , the more prominent is the short duration of action and can not be passed down
Experiments have shown that after cells are transfected with miRNA inhibitors, they can act for up to 7 days, which obviously cannot meet the requirements of some disease research
The inability to be passed on to the next generation restricts some studies on embryonic development

Method used

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  • Method for knocking out microRNA gene family by utilizing CRISPR-Cas9 specificity
  • Method for knocking out microRNA gene family by utilizing CRISPR-Cas9 specificity
  • Method for knocking out microRNA gene family by utilizing CRISPR-Cas9 specificity

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] 1. Construct the Cas9 expression vector in vitro, named pGEM-Cas9, the sequence is as shown in SEQ ID NO.1, and the time is 3 days.

[0035] (1) Primer design

[0036] Using the px330 (Addgene:42330) sequence as a template, the primer Cas9-F (the underlined part is the T7 promoter sequence) and the primer Cas9-R were designed.

[0037] Cas9-F:

[0038] 5'- ATGGACTATAAGGACCACGAC-3';

[0039] Cas9-R: 5'-GCGAGCTCTAGGAATTCTTAC-3'.

[0040] (2) PCR amplification

[0041]Using px330 as a template, PCR reaction was carried out under the action of primer Cas9-F and primer Cas9-R, and the amplified product was analyzed by gel electrophoresis, and the target fragment size was 5309bp, such as figure 2 shown.

[0042] The PCR reaction system is:

[0043]

[0044] The PCR reaction conditions are:

[0045]

[0046] (3) Construction of pGEM-Cas9 expression vector

[0047] Take 1 μl of the PCR product of step (2) and connect it to the pGEM-T vector, and place it at roo...

Embodiment 2

[0076] 1. Design sgRNA for mmu-miR-154 and mmu-miR-382 genes, add a BbsI restriction site (underlined) at the 5' end of the primer, and obtain miR-154 sgRNA (sequence such as SEQ ID NO.3) through denaturation and annealing reactions ) and miR-382sgRNA (sequence such as SEQ ID NO.4), the time is 2 days.

[0077] sgRNA-miR-382-F:5'- TACTTGTGACGAATCATTCA-3'

[0078] sgRNA-miR-382-R:5'- TGAATGATTCGTCACAAGTAC-3'

[0079] sgRNA-miR-154-F:5'- TATTCGTGACGAATCATACA-3'

[0080] sgRNA-miR154-R:5'- TGTATGATTCGTCACGAATAC-3'

[0081] Denaturation, annealing reaction system is as follows:

[0082]

[0083]

[0084] The reaction system in the PCR instrument is as follows: 37°C for 30min; 95°C for 5min; 95-25°C, 5°C / min, and the product is miR-382sgRNA.

[0085] Denaturation, annealing reaction system is as follows:

[0086]

[0087] The reaction system in the PCR instrument was as follows: 37°C, 30min; 95°C, 5min; 95-25°C, 5°C / min, and the product was miR-154sgRNA.

[0...

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Abstract

The invention discloses a method for knocking out a microRNA gene family by utilizing CRISPR-Cas9 specificity, wherein a target gene is knocked out by mainly adopting a CRISPR / Cas9 system. The microRNA family is knocked out by utilizing the specificity of the CRISPR / Cas9 system for the first time; the disadvantage that only one gene can be knocked out for one time in traditional transgenosis can be overcome by utilizing the novel method; the time for establishing a model organism is reduced to three weeks; furthermore, the construction step is simple; an expensive molecular reagent is also reduced; the period for constructing a genetically modified mouse is greatly shortened to four months; multiple genes constructed for one time can be knocked out simultaneously; the fund investment is obviously reduced; F1 generations of mice can be obtained only in need of 50000 Yuan; the gene modification efficiency can be above 90%; the unreliability of the traditional technology is reduced; the operation technology is simple; and a series of complex steps including constructing a targeting vector, screening ES (Embryonic Stem) cells, selectively breeding chimeric mice and the like are unnecessary.

Description

(1) Technical field [0001] The present invention relates to a method for specifically knocking out microRNA genes by CRISPR-Cas9 and the design of specific targeting sgRNA. (2) Background technology [0002] In recent years, my country's pharmaceutical industry has developed rapidly, but at the same time there are many problems, such as weak research and development foundation, flood of imitation of foreign patents, lack of independent intellectual property rights, etc., which seriously restrict the development of my country's pharmaceutical industry. Therefore, it is urgent to enhance my country's drug research and development capabilities. The drug research and development process is the process of disease pathogenic mechanism and drug target screening. Most of the screening process relies on animal disease models of various human diseases, especially mouse models that are closely related to humans (including transgenic and gene knockout) To analyze the pathogenesis of di...

Claims

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Application Information

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IPC IPC(8): C12N15/85A01K67/027
Inventor 黄华荣陈勇龙羊雪芹包美玲曹欢欢张遵义
Owner HANGZHOU NORMAL UNIVERSITY
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