Method for obtaining sheep with different hair colors on basis of CRISPR/Cas9 and sgRNA of targeted ASIP gene

A technology of sheep and genes, applied in the direction of DNA/RNA fragments, other methods of inserting foreign genetic materials, genetic engineering, etc., to achieve the effect of simple construction steps, high specificity, and shortened experimental period

Active Publication Date: 2016-09-21
新疆畜牧科学院生物技术研究所
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  • Abstract
  • Description
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  • Application Information

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Problems solved by technology

In addition, different sheep breeds will have different types of coat c

Method used

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  • Method for obtaining sheep with different hair colors on basis of CRISPR/Cas9 and sgRNA of targeted ASIP gene
  • Method for obtaining sheep with different hair colors on basis of CRISPR/Cas9 and sgRNA of targeted ASIP gene
  • Method for obtaining sheep with different hair colors on basis of CRISPR/Cas9 and sgRNA of targeted ASIP gene

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1, preparation sgRNA and Cas9mRNA

[0036] 1. Design the target sequence of sheep ASIP gene and the sgRNA that recognizes the target sequence

[0037] The full-length sequence of the sheep ASIP gene is shown in GENBANK ACCESSION NO.NC_019470 (VERSIONNC_019470.1; GI:417531912; linear CON 03-APR-2015).

[0038] Sequence 1 of the sequence listing is a partial segment of the sheep ASIP gene, which has three exons (222-381 nucleotides, 795-859 nucleotides, 1222-1398 nucleotides), It encodes the protein shown in Sequence 2 of the Sequence Listing.

[0039] After a lot of pre-tests and verification tests, the 359th-378th nucleotides in sequence 1 in the sequence listing were selected as the target sequence of the sgRNA for the sheep ASIP gene.

[0040] 2. Preparation of sgRNA ASIP

[0041] 1. Use restriction endonuclease BbsI enzyme single digestion px330 plasmid, then carry out 1% agarose gel electrophoresis (the 1% agarose gel electrophoresis figure of restric...

Embodiment 2

[0052] Example 2, Detection of the Mutation Efficiency of Injected sgRNA / Cas9mRNA Embryos

[0053] 1. Obtaining fertilized sheep eggs

[0054] 1. Oocyte maturation

[0055] Collect sheep ovaries from the slaughterhouse (the ovaries are from Kazakh sheep), wash them with normal saline for 3-4 times, extract the oocytes, wash them with maturation solution for 3-4 times, and then drop them into well-balanced maturation solution (maturation solution The volume of the drop is 75-78μl, and each drop is filled with 25-30 oocytes), and placed in a solution containing 5% CO 2 cultured in a 38.6°C incubator (the following incubator cultures are all under the same conditions).

[0056] Equilibrate the mature solution: place the mature solution drop in the incubator for 2h. Maturation solution: TCM199 culture solution + 10% by volume FBS + 0.05IU / ml FSH + 0.05IU / ml LH + 1μg / ml estradiol + 24.2μg / ml sodium pyruvate + 0.1mM / L cysteine +10ng / ml EGF+100IU / ml penicillin+100IU / ml streptomyc...

Embodiment 3

[0077] Example 3, Production of gene-edited sheep

[0078] 1. Selection of experimental sheep

[0079] Xinjiang fine-wool sheep with good body condition, no reproductive diseases and 2-4 years old were selected as donor ewes. Select the Altay sheep with a weight of more than 50kg, an age of 2-4 years, good body condition and no reproductive diseases as recipient ewes. Select Xinjiang fine-wool sheep with a body weight of 70-85kg, excellent semen detection and 1-3 years old as semen collection rams.

[0080] 2. Synchronous estrus and superovulation

[0081] During the estrous cycle of the sheep, the donor ewes were put into the vagina of the CIDR vaginal suppository, and on the 10th day after the CIDR vaginal suppository was put in, FSH (Ningbo Sansheng Company, China) was injected continuously in a decreasing manner, once every 12 hours, for a total of 3 days , the total dose is 240 units / only, take out the CIDR suppository in the morning of the 12th day, wash the vagina, a...

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Abstract

The invention discloses a method for obtaining sheep with different hair colors on the basis of CRISPR/Cas9 and sgRNA of a targeted ASIP gene. The invention provides sgRNA (ASIP-sgRNA) capable of achieving specific and targeted modification of a sheep ASIP gene, and the sgRNA (ASIP-sgRNA) is RNA as shown from the third nucleotide to the 22<nd> nucleotide of 5' tail end of a sequence 4 of a sequence list or RNA with the nucleotides from the third nucleotide to the 22<nd> nucleotide of the 5' tail end of the sequence 4 of the sequence list. The ASIP-sgRNA particularly can be the RNA as shown in the sequence 4 of the sequence list. The invention further provides a method for obtaining sheep with changed hair colors. The method comprises the following steps that co-transfection of sgRNA capable of achieving specific and targeted modification of the sheep ASIP gene and Cas9mRNA on sheep cells is conducted, and therefore the sheep ASIP gene is deleted, and the sheep with the changed hair colors are obtained. According to the method for obtaining the sheep with different hair colors on the basis of CRISPR/Cas9 and sgRNA of the targeted ASIP gene, a CRISPR/Cas9 genome-editing technology is combined with a microinjection technology, and an effective technological means is provided for artificially changing the hair colors of the sheep.

Description

technical field [0001] The invention belongs to the field of animal genetic engineering and relates to CRISPR / Cas9 technology, in particular to a method for obtaining sheep with different coat colors based on the CRISPR / Cas9 gene knockout technology and sgRNA for specifically targeting ASIP gene modification. Background technique [0002] Genome manipulation technology is a cutting-edge technology developed in recent years based on genome and genetic information technology to achieve precise editing of specific genes or genome target sites through artificial design. It has become a research hotspot in the fields of biomedicine, agricultural animal breeding, and model animals. . In the field of animal breeding, in order to breed a variety or establish a specialized strain, it is necessary to improve its production performance and stabilize its genetic basis. In the sheep breeding work, the coat color trait is an important breed characteristic and production trait, which is o...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/12C12N15/55C12N15/89A01K67/027
CPCA01K67/0276A01K2217/075A01K2227/103A01K2267/02C07K14/47C12N9/22C12N15/113C12N15/89C12N2310/10
Inventor 刘明军张雪梅贺三刚李文蓉刘晨曦彭新荣林嘉鹏陈磊
Owner 新疆畜牧科学院生物技术研究所
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