Two sgRNA guide sequences specifically targeting pig Pax7 gene and application

A specific and sequence technology, applied in the field of plant genetic engineering, to achieve the effect of effective modification and mutation

Active Publication Date: 2021-03-16
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the research on Pax7 is mainly concentrated in mice, and there are relatively few studies in mammals such as pigs

Method used

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  • Two sgRNA guide sequences specifically targeting pig Pax7 gene and application
  • Two sgRNA guide sequences specifically targeting pig Pax7 gene and application
  • Two sgRNA guide sequences specifically targeting pig Pax7 gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1 Obtaining of sgRNA sequence

[0057] According to the genome sequence of the pig Pax7 gene (accession number AEMK02000045.1), four Pax7-sgRNAs were designed near its start codon ATG, and CACC bases were added to its 5' end to obtain the Pax7-sgRNA-F primer sequence; at the same time Obtain its corresponding DNA complementary strand according to the guide sequence, and add AAAC bases at the 5' end to obtain the Pax7-sgRNA-R primer sequence; synthesize the above-mentioned Pax7-sgRNA-F and Pax7-sgRNA-R respectively, and synthesize the two The oligonucleotides were denatured at 95°C for 10 minutes and annealed at 65°C for 50 minutes to form double-stranded DNA with cohesive ends. See Table 1 for the specific oligonucleotide sequences.

[0058] Table 1 The oligonucleotide sequences corresponding to the four sgRNAs

[0059]

Embodiment 2

[0060] Example 2 Construction of the vector expressing sgRNA

[0061] 1. Construction of linearized vector: Plasmid vector pX330 (Addgene) was digested with BbsⅠ, the digestion system was 50uL system: BbsⅠ2uL, 10X NEB Buffer 5uL, plasmid 2ug, made up to 50uL with double distilled water. Digestion conditions: enzyme digestion at 37°C for 2 hours; after agarose gel electrophoresis of the digested products, gel recovery was carried out using a gel recovery kit (Omega). See the instructions of the aforementioned kit for the recovery method.

[0062] 2. Ligation: Ligate the recovered pX330 digested with the double-stranded DNA prepared in Example 1. The ligation system is 10uL. Make up to 10uL with double distilled water; the connection condition is 16°C for 2h.

[0063] 3. Transformation: Transform the ligation product into Escherichia coli competent DH5α. The specific transformation method is: take out Escherichia coli competent DH5α at -80°C and dissolve on ice; Put it in a ne...

Embodiment 3 4

[0065] Example 3 The specific targeting verification of four sgRNAs

[0066] 1. Cell culture and collection: PK15 cells (ATCC) were revived with complete medium, and transfected after 2-3 passages. One day before transfection, 5×10 5 ~8×10 5 Cells were seeded into 6-well plates and transfected when the density reached 70%.

[0067] 2. Transfection: For specific steps of transfection, refer to the operation instructions of Lipo2000. The four vectors constructed in Example 2 were transfected with lipo2000 (Invitrogen), and the cells were collected after 48 hours, and the NC plasmid (empty plasmid) was used as a control.

[0068] 3. Screening of specific targeting sgRNA: According to the position of the designed sgRNA sequence, design identification primers, the primer sequence is shown in Table 2, extract cell DNA after 48h, and carry out PCR amplification with identification primers, the reaction system is 50uL: 10XBuffer: 5uL, dNTP: 4uL, Lataq: 0.5uL, F: 1uL, R: 1uL, DNA: 1...

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Abstract

The invention belongs to the technical field of animal gene engineering, and particularly relates to two sgRNA guide sequences specifically targeting a pig Pax7 genes and an application. According tothe invention, four sgRNA guide sequences are designed near an initiation codon ATG of the pig Pax7 gene, and two sgRNAs capable of specifically targeting the vicinity of the initiation codon ATG of the pig Pax7 gene are selected from the four sgRNA guide sequences by using a CRISPR / Cas9 system. By combining the sgRNA sequences provided by the invention with the CRISPR / Cas9 system for use, editingor knockout of the Pax7 gene in pigs can be realized, so that marking or low expression of the Pax7 gene is realized, and a technical foundation is laid for preparation of Pax7 transgenic pigs.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and specifically relates to two sgRNA guide sequences specifically targeting porcine Pax7 gene and application thereof. The invention specifically relates to a sgRNA sequence specifically targeting the vicinity of the initiation codon ATG of the porcine Pax7 gene and an application thereof. Background technique [0002] CRISPR / Cas is an adaptive immune defense formed during the long-term evolution of bacteria and archaea, which can be used to fight against invading viruses and foreign DNA. The CRISPR / Cas9 system is based on the transformation of the class II CRISPR system. In 2012, Jinek verified through in vitro experiments that not only mature crRNA (CRISPR RNA) but also tracrRNA (trans-activating crRNA) are required for the function of Cas9, and that the cleavage site of Cas9 protein has great specificity, both of which are located in PAM ( protospacer adjacent motif) at the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/85C12N15/10C40B50/06
CPCC12N15/113C12N15/85C12N15/1093C40B50/06C07K14/47C12N2310/20C12N2800/107
Inventor 王恒任瑞敏蒋云琪尹彬旭廖胤龙蔡豪朱桂玉
Owner HUAZHONG AGRI UNIV
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