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A method for knocking out the fgf5 gene in animals using the CRISPR-Cas9 system

A technology for knocking out animals and genes, which is applied in the field of animal genetic engineering and genetic modification, can solve the problems of complicated operation, complicated design and production of ZFNs, and high cost, and achieve the effects of improving transfection efficiency, wide applicability, and simple operation

Active Publication Date: 2019-05-21
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Although ZFNs and TALENs have the advantages of high targeting efficiency compared with traditional homologous recombination, they still have many defects, mainly including: 1. The DNA cutting domain of ZFNs and TALENs is FolkI, which must form a dimer Therefore, at least two DNA expression structures must be used for gene targeting in mammals, which will have higher requirements for transfection efficiency during cell transfection; 2. The design and production of ZFNs are relatively complicated and costly High, and it is difficult to control the cost within an acceptable range when applied to a large number of mammalian gene targeting; 3. The DNA recognition rules and design requirements of ZFNs and TALENs are relatively strict, and it may not be possible to find suitable ZFNs and TALENs recognition regions in the target gene sequence , so that it cannot be used for gene targeting; 4. When targeting different genes or the same gene of different species, it is necessary to design and construct new ZFNs and TALENs expression plasmids or mRNAs, and the operation is complicated; 5. ZFNs and When TALENs perform multi-gene targeting, it is difficult to obtain high targeting efficiency due to the limitation of the size of the carrier and mRNA molecules
There is no report on the knockout of animal FGF5 gene by CRISPR-Cas9 expression system

Method used

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  • A method for knocking out the fgf5 gene in animals using the CRISPR-Cas9 system
  • A method for knocking out the fgf5 gene in animals using the CRISPR-Cas9 system

Examples

Experimental program
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Effect test

Embodiment 1

[0034] Example 1 Construction of the CRISPR-Cas9 expression system for the FGF5 gene

[0035] 1. Compared the FGF5 gene sequences of different species (human, mouse, pig, cow, sheep, goat), found a relatively conserved region, designed sgRNA in these two regions and obtained a sgRNA sequence information. The DNA sequence of the sgRNA specifically targeting the second exon of the FGF5 gene is shown in SEQ ID NO.1.

[0036] 2. Construction of pX330-F2: (1) Design and synthesize the DNA sequence of the sgRNA recognition region that recognizes the second exon of FGF5, as shown in SEQ ID NO.1; (2) Gradient after phosphorylation of the synthesized sgRNA sequence Cooling and annealing, the specific steps are to mix the synthesized oligoDNA with 10X T4Ligation Buffer and T4PNK at a ratio of 2:2:1, then add 3 times the volume of water to make up the system, then incubate at 37°C for 30min, then denature at 95°C for 5min, Afterwards, the temperature was lowered to 25°C at a rate of 5°...

Embodiment 2

[0040] Example 2 In vitro transcription

[0041] Use the constructed in vitro transcription vectors pIVT-F2-T and pCas9-puro3 to perform in vitro transcription mediated by the T7 promoter, that is, use the T7 promoter as the promoter for in vitro transcription, and use RNA polymerase to realize the conversion from DNA to mRNA in vitro For the transcription process, the specific method is: use SalI and NotI to linearize the vectors pIVT-M2-T and pCas9-puro3 respectively, then use the linearized in vitro transcription vector as a template, add T7 transcriptase, buffer and rNTPs, and incubate at 37°C for 6h. Then add DNase at 37°C for 15 minutes to digest and remove the template DNA, then use phenolform extraction to remove protein impurities, ethanol precipitation to obtain the transcribed mRNA, and purify the transcribed mRNA with an adsorption column. The specific method is: add 3.5 Double the volume of binding buffer and 2.5 times the volume of absolute ethanol, mix well and ...

Embodiment 3

[0042] Example 3 Production of gene-targeted mice using the CRISPR-Cas9 system mRNA targeting the FGF5 gene

[0043] 1. Pronuclear injection and embryo transfer

[0044] The pronuclear fertilized eggs of B6D2F1 mice were taken, and the premixed Cas9mRNA / sgRNA mixture (the final concentration of Cas9mRNA was 150ng / μl, and the final concentration of sgRNA was 20ng / μl) was injected into the cytoplasm or in the nucleus. After the injection, the fertilized eggs are transferred to the culture medium for short-term culture, and then transplanted into the oviduct of recipient mother mice to produce gene-targeted mice.

[0045] 2. Identification of gene targeting mice

[0046] After the birth of the surrogate mother mouse, cut off about 1 cm of mouse tail when the offspring grow to 2 weeks old, digest with proteinase K at 55°C, and extract the mouse tail genome by phenolform extraction. Using the mouse tail genome as a template, design primers targeting the second exon of FGF5, ampl...

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Abstract

The invention provides a method for knocking off an animal FGF5 gene by using a CRISPR-Cas9 system. The method comprises the following steps: firstly, acquiring a DNA sequence aiming at an sgRNA recognition area of a second FGF5 exon, wherein the base sequence of the DNA sequence is as shown in SEQ ID NO.1; secondly, establishing an sgRNA expression structure of the second FGF5 exon, inserting a T7 starter before an sgRNA transcriptional start site, establishing an in-vitro transcription carrier of Cas9 protein, and regulating and controlling by using the T7 starter. Cas9 mRNA and sgRNA are obtained through the in-vitro transcription carrier of Cas9 and sgRNA, and the method can be used for knocking off the animal FGF5 gene.

Description

technical field [0001] The invention belongs to the field of animal genetic engineering and genetic modification, and in particular relates to a method for knocking out animal FGF5 gene by using a CRISPR-Cas9 expression system. Background technique [0002] Since the rise of genetic engineering in the 1980s, a large number of gene editing technologies have emerged to meet the needs of scientific research. Among them, gene targeting technology is a technology for targeted and fine modification of genes in higher animals. Traditional gene targeting technology relies on spontaneous homologous recombination (HR, homologous recombination) in vivo, and the efficiency is only about 1 / 10 6 . In recent years, in order to solve the problem of low efficiency of homologous recombination, people use artificially constructed hybrid molecules to cut specific DNA sequences to improve the efficiency of gene targeting. Among them, artificial compound molecules with endonuclease as the core a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/85C12N15/66C12N15/89A01K67/027
Inventor 侯健厉建伟安晓荣
Owner CHINA AGRI UNIV
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