Preparation method of staphylococcus aureus CRISPR/Cas9 system and application of system in constructing mouse model

A staphylococcus aureus and aureus technology, applied in the field of molecular biology, can solve the problems of unclear gene editing and restrict the application of Staphylococcus aureus CRISPR/Cas9 system, and achieve good application prospects

Inactive Publication Date: 2016-09-21
GUANGZHOU MAGIGEN BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, whether the Staphylococcus aureus CRISPR / Cas9 system can perform gene editing in fertilized mouse eggs is still unclear, and the preparation method of Cas9 mRNA and gRNA for the components of the Staphylococcus aureus CRISPR / Cas9 system has not yet been established, which severely restricts Application of Staphylococcus aureus CRISPR / Cas9 system in the construction of genetically modified mouse model

Method used

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  • Preparation method of staphylococcus aureus CRISPR/Cas9 system and application of system in constructing mouse model
  • Preparation method of staphylococcus aureus CRISPR/Cas9 system and application of system in constructing mouse model
  • Preparation method of staphylococcus aureus CRISPR/Cas9 system and application of system in constructing mouse model

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Staphylococcus aureus CRISPR / Cas9 system components Cas9 mRNA and gRNA preparation of

[0040] 1. Preparation of Cas9 mRNA and gRNA

[0041] (1) The preparation method of the Cas9 mRNA is: (1) preparing the transcription template DNA of Staphylococcus aureus Cas9 containing a T7 promoter; (2) preparing the Cas9 mRNA of Staphylococcus aureus;

[0042] More specifically, preferably, the method for preparing the transcription template DNA of Staphylococcus aureus Cas9 comprising the T7 promoter in step (1) is: using a synthetic Cas9 upstream primer and a Cas9 downstream primer to generate a plasmid containing the Cas9 coding DNA sequence of Staphylococcus aureus PX601 (purchased from Addgene) was used as a template, and the transcription template DNA of Staphylococcus aureus containing T7 promoter was amplified by PCR; the PCR product was purified by column purification with PCR product purification kit (Axygen), and then used different Elution...

Embodiment 2

[0091] Example 2 Staphylococcus aureus CRISPR / Cas9 system mediated Tyr single gene knockout

[0092] 1. In order to use Staphylococcus aureus CRISPR / Cas9 system to achieve single gene knockout in mouse fertilized eggs, we targeted Tyr Gene designed a gRNA. Tyr The knockout of the gene will change the coat color of the pups from black to white, which is conducive to the rapid identification of knockout mice. First, we transcribed Tyr gRNA, and then Tyr gRNA (50ng / μl) was mixed with Staphylococcus aureus Cas9 mRNA (100 ng / μl) and injected into 0.5-day-old mouse fertilized eggs. After the injection, the fertilized eggs of the mice were transplanted into the fallopian tubes of 0.5-day-old pseudopregnant mice. After 20 days, the pseudo-pregnant mice would give birth to pups. By amplifying the target site by PCR, and then using T7 endonuclease I to cut the PCR product (T7E1 experiment), we found that 81.8% (9 / 11) of the mice were Tyr Gene knockout mice. ...

Embodiment 3

[0096] Example 3 Staphylococcus aureus CRISPR / Cas9 system mediated Slx2 ， ZP1 ， Tyr triple knockout

[0097] 1. In order to use the Staphylococcus aureus CRISPR / Cas9 system to simultaneously knock out multiple genes in mouse fertilized eggs, we designed Slx2 , ZP1 , Tyr Gene gRNA. Then these three gRNAs (each with a concentration of 50 ng / μl) were mixed with Staphylococcus aureus Cas9 mRNA (100 ng / μl) and injected into 0.5-day-old mouse fertilized eggs. Then, these mouse fertilized eggs were cultured for 3.5 days, and then the genomic DNA of individual embryos were amplified by whole genome amplification method. Then, use the amplified product as a template to amplify by PCR Slx2 , ZP1 , Tyr Target site, and using T7 endonuclease Ⅰ to cut the PCR product, we found that the Staphylococcus aureus CRISPR / Cas9 system can efficiently knock out three genes in mouse fertilized eggs (46.7%, 14 / 30).

[0098] 2. The results are attached Figu...

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Abstract

The invention discloses a preparation method of a staphylococcus aureus CRISPR / Cas9 system and an application of the system in constructing a genetically modified mouse model. The staphylococcus aureus CRISPR / Cas9 system is composed of two components, namely Cas9 mRNA and gRNA, wherein a preparation method of the Cas9 mRNA is achieved by adding a T7 promoter to the upstream region of original Cas9 coding DNA, and a preparation method of the gRNA is achieved by adding the T7 promoter to the upstream region of an original gRNA coding sequence. The staphylococcus aureus Cas9 mRNA and gRNA, which are injected to mouse fertilized embryos through micro-injection, can achieve gene editing and modification of various types, such as single-gene knockout, multi-gene knockout and / or gene knock-in and the like, on the mouse fertilized embryos; therefore, the CRISPR / Cas9 system has a good application prospect in the aspects of fertilized embryo gene editing and modification of such animals as mouse and the like as well as construction of animal models.

Description

technical field [0001] The invention belongs to the technical field of molecular biology. More specifically, it relates to the preparation of Staphylococcus aureus CRISPR / Cas9 system components and its application in the construction of genetically modified mouse models. Background technique [0002] The completion of the Human Genome Project in 2003 set off an upsurge in gene function research, making functional genomics the focus of life science research. Mice have become an important tool for analyzing human gene functions due to their high similarity to humans in anatomical structure, physiology, and genome. In the late 1980s, the emergence of mouse gene knockout technology enabled us to precisely modify the mouse genome for the first time, and use mice to construct human disease models, which provided the best way to elucidate the pathogenesis, prevention and treatment of diseases. strong evidence. Especially for the modification of oncogenes, it has greatly promoted...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/55C12N15/113C12N15/10C12N15/89A01K67/027
CPCC12N9/22A01K67/0275A01K2227/105A01K2267/03C12N15/10C12N15/113C12N15/89C12N2310/10
Inventor 黄军就梁普平松阳洲张曦亚
Owner GUANGZHOU MAGIGEN BIOTECH
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