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Method for acquiring gene editing sheep by RNA-mediated specific double-gene knockout and special sgRNA for method

A breed-specific, sheep-based technology, applied in DNA/RNA fragments, recombinant DNA technology, artificial cell constructs, etc., can solve the high cost and technical requirements of large animal genome modification and transformation, the difficulty of cultivating new varieties, and the long breeding cycle to avoid biosafety issues, shorten the experimental cycle, and reduce the cost of the experiment

Active Publication Date: 2015-12-09
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

However, the conventional breeding cycle is long, the predictability is poor, and the selection efficiency is low. The genetic gain obtained by conventional breeding methods in variety improvement is gradually flattening, especially in the breeding of varieties with multiple excellent traits such as high yield, high quality, and stress resistance. It is becoming more and more difficult to breed new species, and the cost and technical requirements for modification and transformation of large animal genomes are still high

Method used

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  • Method for acquiring gene editing sheep by RNA-mediated specific double-gene knockout and special sgRNA for method
  • Method for acquiring gene editing sheep by RNA-mediated specific double-gene knockout and special sgRNA for method
  • Method for acquiring gene editing sheep by RNA-mediated specific double-gene knockout and special sgRNA for method

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Experimental program
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Effect test

Embodiment 1

[0042] Embodiment 1, preparation sgRNA and Cas9mRNA

[0043] 1. Design the target sequence and sgRNA that recognizes the target sequence

[0044] 1. Design the target sequence of sheep MSTN gene and the sgRNA that recognizes the target sequence

[0045] The partial sequence of the sheep MSTN gene is shown in Sequence 1 of the Sequence Listing. The 1st to 3rd nucleotides from the 5' end are start codons, the 4989th to 4991st nucleotides are stop codons, and the 4611st to 4991st nucleotides are Nucleotides are exon 3. The protein encoded by the sheep MSTN gene is shown in sequence 2 of the sequence listing.

[0046] Six sgRNAs were designed for the target sequence of the sheep MSTN gene (the target sequence is on exon 3 of the sheep MSTN gene), see the italic parts in MSTN-TF1 to MSTN-TF6 in Table 1.

[0047] 2. Design the target sequence of sheep FGF5 gene and the sgRNA that recognizes the target sequence

[0048] The full-length sequence of the sheep FGF5 gene is shown in ...

Embodiment 2

[0080] Example 2, sgRNA / Cas9mRNA mutation efficiency detection

[0081] 1. Obtaining fertilized sheep eggs

[0082] 1. Oocyte maturation

[0083] 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).

[0084] Equilibrate the mature solution: place the mature solution drop in the incubator for 2h. Maturation solution: TCM199 culture solution + 10% FBS by volume + 0.05IU / mlFSH+0.05IU / mlLH+1μg / mlestradiol+24.2μg / ml sodium pyruvate+0.1mM / L cysteine+10ng / mlEGF+100IU / ml penicillin+100IU / ml streptomycin.

[0085] 2. In vitro fertilization...

Embodiment 3

[0111] Example 3, Production of gene-edited sheep

[0112] 1. Selection of experimental sheep

[0113] 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 purebred Xinjiang fine-wool sheep with a body weight of 70-85kg, excellent semen detection and 1-3 years old as semen collection rams.

[0114] 2. Synchronous estrus and superovulation

[0115] 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 ...

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Abstract

The invention discloses a method for acquiring gene editing sheep by RNA-mediated specific double-gene knockout and special sgRNA for the method. The sgRNA combination consists of sgRNAMSTN-1 and sgRNAFGF5-1, wherein sgRNAMSTN-1 is sgRNA which can realize specific targeted modification of sheep MSTN gene and is RNA shown in the second to 21st nucleotides in a sequence 6 or RNA with the second to 21st nucleotides of the sequence 6; sgRNAFGF5-1 is sgRNA which can realize specific targeted modification of sheep FGF5 gene and is RNA shown in the second to the 21st nucleotides in a sequence 8 or RNA with the second to 21st nucleotides of the sequence 8. According to the method for acquiring gene editing sheep by RNA-mediated specific double-gene knockout and special sgRNA for the method, the CRISPR / Cas9 genome editing technology and the micro-injection technology are combined, so that the sheep targeting efficiency is higher and more accurate, sheep double-gene knockout is realized for the first time in the generation, improvement on sheep meat production and wool production is greatly promoted, and a larger space and a more effective technical tool are provided for breeding of new sheep varieties.

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 gene-edited sheep by RNA-mediated specific knockout of double genes and a special sgRNA thereof. 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, since the potential of traditional breeding methods to increase production has reached its limit, using efficient and stable genome manipulation technology to innovate breeding methods and improve the efficiency and technical level of modern animal breeding is crucial for the creation of new breeding materials and ...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/11C12N5/071
Inventor 刘明军张雪梅彭新荣吴阳升林嘉鹏刘晨曦贺三刚李文蓉
Owner 新疆畜牧科学院生物技术研究所
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