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Method for improving CRISPR/Cas9 mediated biallelic mutation efficiency and application thereof

An allelic and efficient technology, applied in the fields of genetic engineering and molecular genetics and breeding, can solve problems such as the acquisition of target traits that affect the efficiency of animal gene editing

Active Publication Date: 2020-10-20
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The so-called "mosaic phenomenon" refers to the fact that when the CRISPR / Cas9 system is applied to the gene editing of fertilized eggs of multicellular organisms, since the fertilized eggs will split into different blastomeres, the editing ability and repair method of Cas9 protein on different blastomeres may be different, leading to the emergence of chimeric individuals with both edited cells and unedited cells, which in turn affects the efficiency of gene editing in animals and the acquisition of target traits in animals

Method used

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  • Method for improving CRISPR/Cas9 mediated biallelic mutation efficiency and application thereof
  • Method for improving CRISPR/Cas9 mediated biallelic mutation efficiency and application thereof
  • Method for improving CRISPR/Cas9 mediated biallelic mutation efficiency and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Design of sgRNA for gene editing of MSTN and FGF5

[0039] In this example, the MSTN and FGF5 genes of sheep are taken as examples to design sgRNA for gene editing by CRISPR / Cas9.

[0040] In order to achieve high gene editing efficiency, the design principles of sgRNA are as follows: the length of the sequence complementary to the genome sequence is 20-30nt, there must be a PAM (NGG) sequence at the 3' end of the sequence, and SNP and polyT sequences should be avoided. It is best to start with the base "G".

[0041] Two targeting sites were designed for the MSTN gene of sheep, both located on the third exon of the MSTN gene. The arrangement and structure of the two targets in the MSTN genome are as follows: figure 1 As shown, named as Cr1 and Cr2, figure 1The corresponding targeting sequence in the genome and the PAM segment of the site are shown. The gray sequence is the crRNA sequence corresponding to the sgRNA and the target site, followed by tracrRNA t...

Embodiment 2

[0043] Example 2 In vitro transcription of Cas9 mRNA and sgRNA

[0044] The Q5 high-fidelity DNA polymerase was used to amplify the Cas9 (Cas9 gene sequence shown in SEQ ID NO.4) and the sgRNA sequence containing the T7 promoter, respectively, and the template for in vitro transcription was obtained after purification. T7-Cas9 was transcribed with mMESSAGE mMachineT7Ultra Kit, and T7-sgRNA was transcribed with MEGAshortscript Kit. The transcribed Cas9 RNA was treated with RNA-specific loading and became a single-stranded straight line. After 2% agarose-TBE gel electrophoresis, it was shown that Cas9 was capped and transcribed at about 4200 nt. The post-transcribed Cas9 mRNA band was clear and the size was correct (such as image 3 shown in A). After the transcription of sgRNA (about 100nt) was purified and recovered, it was detected by agarose gel electrophoresis. The sgRNA band was clear and the size was correct (such as image 3 shown in B).

Embodiment 3

[0045] Example 3 Using the CRISPR / Cas9 system to edit the MSTN and FGF5 genes of sheep

[0046] In this example, the prokaryotic microinjection method was used to introduce the Cas9 mRNA synthesized by in vitro transcription and sgRNA of MSTN and FGF5 genes in Example 2 into sheep prokaryotic embryos to achieve gene knockout of MSTN and FGF5.

[0047] The Cas9 mRNA and sgRNA synthesized by in vitro transcription in Example 2 were mixed to prepare a gene editing nucleic acid solution for microinjection. According to the different injection ratios of Cas9 mRNA and sgRNA, sheep pronuclear stage embryos were divided into three groups: (1) Control group: the molar concentration ratio of Cas9 mRNA and sgRNA was 1:2, two microinjections were performed, and the first microinjection The concentrations of Cas9 mRNA and sgRNA were 5.23*10 -9 mol / l and 10.46*10 -9 mol / l, the concentrations of Cas9 mRNA and sgRNA in the second microinjection were 7.93*10 -9 mol / l and 15.86*10 -9 mol / l,...

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Abstract

The invention relates to the technical field of genetic engineering and molecular genetic breeding, in particular to a method for improving the CRISPR / Cas9 mediated biallelic mutation efficiency and an application thereof. A CRISPR / Cas9 gene editing method provided by the invention comprises the following steps of introducing gene editing nucleic acid into a prokaryotic embryo, wherein the gene editing nucleic acid comprises Cas9 mRNA and sgRNA, and in the gene editing nucleic acid, the molar concentration ratio of the Cas9 mRNA to the sgRNA is 1:10 to 1:20. According to the gene editing method provided by the invention, the efficiency of biallelic mutation is remarkably improved, so that the proportion of gene editing chimeric individuals is effectively reduced, and the proportion of biallelic mutation individuals is improved; and the gene editing method has important application value for construction and genetic breeding of animals with target traits.

Description

technical field [0001] The invention relates to the technical fields of genetic engineering and molecular genetic breeding, in particular to a method for improving the efficiency of biallelic mutation mediated by CRISPR / Cas9 and its application. Background technique [0002] CRISPR / Cas9 technology is an RNA-mediated genome editing technology, which can precisely knock out, knock in and replace genes, so as to realize the purpose of studying gene function, removing or repairing targeted genes. [0003] The CRISPR / Cas9 system mainly consists of two parts: sgRNA and Cas9 protein. sgRNA is composed of CRISPR RNA (crRNA) and trans-activating crRNA (trans-activating crRNA, tracrRNA), crRNA includes a sequence that can pair with tracrRNA to form a double-stranded RNA structure, and also includes a sequence that can be complementary to the target region of the target gene, and This identifies the target sequence. In practical applications, crRNA and tracr RNA are often chiseled in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/90C12N15/10A01K67/027
CPCC12N15/907C12N15/102A01K67/0275A01K2217/07A01K2227/103A01K2267/02
Inventor 连正兴李岩邓守龙刘国世连玲
Owner CHINA AGRI UNIV
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