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A sgRNA targeted to knock out the sst gene and its CRISPR/Cas9 system and application

A genetic and targeting technology, applied in DNA/RNA fragments, applications, genetic engineering, etc., can solve problems such as low efficiency, cumbersome steps, and long time-consuming

Active Publication Date: 2022-01-07
武汉纽利特生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the CRISPR-Cas9 system is widely used as a gene editing tool. It overcomes the shortcomings of traditional gene editing technology such as cumbersome steps, long time-consuming, and low efficiency. gene editing needs
However, the deletion effect of the CRISPR-Cas9 system in the prior art needs to be further improved

Method used

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  • A sgRNA targeted to knock out the sst gene and its CRISPR/Cas9 system and application
  • A sgRNA targeted to knock out the sst gene and its CRISPR/Cas9 system and application
  • A sgRNA targeted to knock out the sst gene and its CRISPR/Cas9 system and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Construction of Cas9 sgRNA knockout plasmid

[0048] 1.1 Screening and synthesis of sgRNA

[0049] Select the first exon of the porcine SST gene (GenBank accession number: NC_010455.5) as the targeting region, and use the website (http: / / crispor.tefor.net) to design sgRNA for the targeting region. The design conditions are: ① The length of the sgRNA is 20nt, the core sequence is NNNNNNNNNNNNNNNNNNNN(20)-NGG (SEQ ID No.6); ② Add CACCG to the 5' end of the sense strand template, and add AAAC to the 5' end of the antisense strand template to make the oligonucleotide chain and the vector plasmid The cohesive ends are complementary. Finally, two suitable targets were selected and named as SST-sgRNA1 and SST-sgRNA2. CACC was added to the 5' end of the coding strand template, and AAAC was added to the 3' end of the non-coding strand template to complement the cohesive ends formed after digestion with BbsI, and two pairs of CRISPR oligonucleotide chains were designed.

[005...

Embodiment 2

[0053] vector construction

[0054] 1.2.1 Use BbsI to digest the pSpCas9(BB)-2A-Puro plasmid, and the digestion reaction system is as follows:

[0055] Element Amount added pSpCas9(BB)-2A-Puro plasmid 1μg BbsI enzyme 2μL FDbuffer 2μL wxya 2 o

to 50μL Total 50μL

[0056] Operated on ice, the above components were added sequentially, after thorough mixing, enzyme digestion was carried out in a circulating water bath at 37°C for 2 hours.

[0057] 1.2.2 Recover the above digested products according to the instructions of Novizan Gel Recovery Kit.

[0058] 1.2.3 sgRNA oligo annealing and double strand formation

[0059] Dilute each pair of oligonucleotides synthesized by the company to 10 μmol / L and mix according to the following ratio: 16 μL ddH 2 O, 2 μL 10×NEB Buffer 3, 1 μL sense strand, 1 μL antisense strand, denature at 95°C for 5 minutes after mixing, and then naturally anneal at room temperature for 1 hour to form do...

Embodiment 3

[0065] Transfection of porcine kidney fibroblasts by electroporation

[0066] 1.1 Main reagents and materials

[0067] Male Large White piglets aged 1-3 days were from the experimental pig farm of the Institute of Animal Husbandry and Veterinary Medicine, Hubei Academy of Agricultural Sciences; G418 antibiotics were purchased from Sigma; DMEM, DPBS, fetal bovine serum, DMSO and other cell culture and cryopreservation reagents were purchased from Gibco. Electrotransfer buffer was prepared by the laboratory itself.

[0068] 1.2 Isolation and culture of porcine kidney fibroblasts

[0069] Take a newborn 1-3d male large white pig, deeply anesthetized by intraperitoneal injection of pentobarbital sodium (50 mg); fully clean and disinfect the large white pig body surface with 75% alcohol, and then use sterilized medical surgical instruments to remove the double The side kidneys were soaked in 75% alcohol and moved to a clean bench.

[0070] The kidney was washed repeatedly with D...

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Abstract

The invention provides a sgRNA targeting knockout SST gene and its CRISPR / Cas9 system and application, belonging to the technical field of gene knockout. The present invention provides a sgRNA targeted to knock out the SST gene, including SST sgRNA1 and SST sgRNA2, and also provides a CRISPR / Cas9 system comprising the sgRNA. The CRISPR / Cas9 system is transfected into porcine kidney fibroblasts, and the positive cells obtained by screening are obtained to obtain a higher gene deletion efficiency. At the same time, the CRISPR / Cas9 system can improve the efficiency of gene deletion by completely knocking out the pig SST gene. The growth rate of pigs, thereby improving the economic benefits of pig farming, and the CRISPR / Cas9 system is of great significance for further research on the function of the SST gene and its correlation with certain diseases.

Description

technical field [0001] The invention belongs to the technical field of gene knockout, and in particular relates to a sgRNA targeting knockout of SST gene and its CRISPR / Cas9 system and application. Background technique [0002] The SST gene is located on pig chromosome 13, with a length of 1183bp and two exons. It is generally believed that the main function of SST in mammals is to regulate the growth of animals by directly or indirectly inhibiting the secretion of growth hormone. Some measures are often adopted in animal husbandry to reduce the SST content in animals, weaken or eliminate the inhibition of SST on the digestive system, and release the inhibition of SST on growth hormone and insulin-like growth factor 1, thereby increasing the growth rate and body size of animals. But these suppression measures are not suitable for fundamental long-term suppression studies. [0003] The CRISPR-Cas9 gene editing technology uses a short single-stranded guide RNA (single guide ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/85C12N15/90C12N15/12C12N5/10C12Q1/6858
CPCC12N15/113C12N15/85C12N15/907C07K14/4703C12N5/0686C12Q1/6858C12N2310/20C12N2800/107C12N2510/00A01K2217/075A01K2227/108A01K2267/02C12Q2531/113
Inventor 任红艳毕延震王紫君华再东肖红卫朱喆张立苹
Owner 武汉纽利特生物技术有限公司
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