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Method suitable for knocking out Mstn gene of fertilized egg of culter alburnus and application

A technology of gene knockout and cichlid, which is applied in the field of gene editing, can solve the problems of slow growth, miniaturization and underexploitation of cichlid, and achieve the effect of improving the efficiency of gene editing

Active Publication Date: 2021-10-01
ZHEJIANG INST OF FRESH WATER FISHERIES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, most of the cultured Cichlids are wild species that have not been selected for breeding, and their potential genetic advantages have not been fully exploited. In addition, blind breeding, introduction, hybridization and repeated generation breeding have resulted in slow growth, small Germplasm decline or hybridization, germplasm improvement has become an urgent need for the sustainable and healthy development of the Cichlid aquaculture industry

Method used

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  • Method suitable for knocking out Mstn gene of fertilized egg of culter alburnus and application
  • Method suitable for knocking out Mstn gene of fertilized egg of culter alburnus and application
  • Method suitable for knocking out Mstn gene of fertilized egg of culter alburnus and application

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Experimental program
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Effect test

Embodiment 1

[0034] Knockout of Mstn Gene in Fertilized Eggs of C.

[0035] The technological route of gene editing and mutation detection of Cichlid chinensis is as follows: figure 1 shown.

[0036] 1. Design and synthesis of CRISPR system

[0037] 1. Design and synthesis of gRNA

[0038] The gRNA sequence is shown in SEQ ID NO.1. The region where the designed gRNA sequence is located such as figure 2 As shown, the final gRNA selected as image 3 shown.

[0039] 2. Determination of the delivery form and injection concentration of gene editing elements

[0040] The gRNA sequence powder synthesized by the gene company was concentrated according to the instructions. The Cas9 protein used for injection was directly purchased from Thermo Fisher (A36497, 1mg / mL). The delivery form of the gene editing element was in the form of RNP. The final concentration of the Mstn gene knockout reagent microinjection system was 200 ng / μL of Cas9 protein, 100 ng / μL of sgRNA and 0.05 wt% of phenol red ...

Embodiment 2

[0047] The differences between this embodiment and Embodiment 1 are:

[0048] The final concentration of the microinjection system of the knockout reagent for the Mstn gene of the fertilized eggs of Cichlidus chinensis used was 210 ng / μL of Cas9 protein, 110 ng / μL of sgRNA and 0.1 wt% of phenol red indicator.

[0049] The injection volume when performing microinjection was 2.5 nL.

Embodiment 3

[0051] The differences between this embodiment and Embodiment 1 are:

[0052] The final concentration of the microinjection system of the knockout reagent for the Mstn gene of the fertilized eggs of C. chinensis used was 200 ng / μL of Cas9 protein, 100 ng / μL of sgRNA, 100 ng / μL of stachydrine and 0.05 wt% of phenol red indicator.

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Abstract

The invention discloses a method suitable for knocking out an Mstn gene of a fertilized egg of culter alburnus and application, and belongs to the technical field of gene editing. The method includes the steps that a knockout reagent is injected into an embryo of the fertilized egg of culter alburnus through a microinjection method to obtain an Mstn gene deletion type culter alburnus model, wherein the knockout reagent comprises sgRNA, Cas9 protein and a phenol red indicator, and the sequence of the sgRNA is shown as SEQ ID NO.1. According to the method, Cas9 protein and sgRNA are directly delivered into fertilized eggs of culter alburnus through microinjection in an RNP form, and the gene editing efficiency is improved. The microinjection method can bypass multiple layers of barriers such as extracellular matrixes, cell membranes and cytoplasm, sgRNA and Cas9 protein are accurately delivered into cell nucleuses or cytoplasm in a most direct mode, and therefore high control over gene editing can be achieved, and the off-target effect is greatly reduced.

Description

technical field [0001] The invention relates to the technical field of gene editing, in particular to a method and an application suitable for knocking out the Mstn gene of fertilized eggs of C. chinensis. Background technique [0002] CRISPR / Cas9 gene editing technology is a Cas9 nuclease-targeted editing technology guided by sgRNA. It is simple, fast, and low-cost, and has been widely used in gene knockout, insertion, and knockout. Since the first application of gene editing in eukaryotic cells in 2013, it has been developing at a rapid pace. It is not only widely used in the study of gene function and the treatment of diseases, but also in the rapid preparation of mutant individuals in model organisms, the study of the mechanism of growth and development and the laws of genetics, and the improvement of excellent traits of important economic species. [0003] Culter alburnus Basilewsky (Culter alburnus Basilewsky) is one of the important freshwater economic fishes in my c...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N9/22C12N15/89C12N5/10
CPCC12N15/1136C12N9/22C12N15/89C12N5/0604C07K14/495C12N2310/20Y02A50/30
Inventor 郑建波陈乐然贾永义
Owner ZHEJIANG INST OF FRESH WATER FISHERIES
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