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A pair of sgRNAs targeting the porcine rela gene

One gene, one pair technology, applied in the field of sgRNA targeting recognition of porcine RelA gene

Inactive Publication Date: 2018-09-14
青岛市畜牧兽医研究所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, subsequent studies have shown that Cas9 has obvious off-target effects (High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nature Biotechnology. Fu et al, 2013; High-throughput profiling of off-target DNA cleavage reveals RNA -programmed Cas9 nuclease specificity. Nature Biotechnology. Pattanayak et al, 2013)

Method used

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  • A pair of sgRNAs targeting the porcine rela gene
  • A pair of sgRNAs targeting the porcine rela gene
  • A pair of sgRNAs targeting the porcine rela gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Embodiment 1, the construction of sgRNA expression plasmid pair

[0021] 1. sgRNA target design

[0022] The coding region (959-1033, as shown below) of the tenth exon of the porcine RelA gene was extracted from the sequence with the NCBI accession number NM_001114281.1, using the MIT online software (http: / / crispr.mit.edu / ) Design the target site.

[0023] GACCCACCGACCCCCC GGCCTGCAAC CCGGCGCATT GCTGTGCCTT CCCGCAGCTCAGCTTCCGTC CCCAAGCCAG

[0024] The forward target sequence is TCAGCTTCCGTCCCCAAGCC (54-73) and the reverse target sequence is GGAAGGCACAGCAATGCGCC (28-47). The two target sequences are arranged in a "head-to-head" manner, and the distance between the two is 6 bp, that is, there is a 6 bp interval. Name them as forward target T1 and reverse target T2 respectively. target design as figure 1 shown.

[0025] 2. Construction of sgRNA expression plasmid pair

[0026] First, according to the designed target sequence, send it to Beijing Liuhe Huada Gene Tech...

Embodiment 2

[0032] Example 2. Verifying the biological activity of the sgRNA expression plasmid pair constructed in Example 1 by sequencing

[0033] PEF cells (porcine fetal fibroblasts): PEF cells were isolated from aborted pig fetuses (see references for the isolation method: Li Hong, Wei Hongjiang, Xu Chengsheng, Wang Xia, Qing Yubo, Zeng Yangzhi; Banna Miniature Pig Inbred Line The establishment of fetal fibroblast cell line and its biological characteristics; Journal of Hunan Agricultural University (Natural Science Edition); Vol. 36 No. 6; December 2010; 678-682).

[0034] 1. Co-transfect 4 μg of each of the recombinant plasmids pX335-sgRNA-F and pX335-sgRNA-R into 1×10 6PEF cells to obtain recombinant cells. The specific steps of transfection are as follows: use a nuclear transfer instrument (Amaxa, model: AAD-1001S) and a matching mammalian fibroblast transfection kit (Amaxa, product number: VPI-1002) for transfection. First, use 0.05% trypsin (Gibco, catalog number: 610-5300AG)...

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Abstract

The invention discloses sgRNAs targeting a pig RelA gene. The sgRNA pair comprises sgRNA-F and sgRNA-R. Each of the sgRNA-F and sgRNA-R comprises 102 nucleotides. A RNA fragment comprising 2th-21th sits of the tail end 5' can identify a pig chromosome RelA gene. A RNA fragment comprising 22th-102th sites is a skeleton RNA fragment capable for bonding with Cas9n nuclease. The sgRNA-F is shown in the sequence 2 in the sequence table. The sgRNA-R is shown in the sequence 4 in the sequence table. Through a Cas9n system, in a cell or individual level, the pig RelA gene is knocked out or modified so that pig RelA gene functions are analyzed and a pig RelA gene mutant library is constructed for pig breeding service.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a pair of sgRNA targeting recognition of porcine RelA gene and application thereof. Background technique [0002] ZFN and TALEN targeting technologies are two relatively mature site-directed mutagenesis technologies, but the construction procedures of these two technologies are relatively cumbersome, and a pair of corresponding nucleases needs to be constructed for each site. The CRISPR / Cas9 system’s recognition of specific sites is guided by small crRNAs. The CRISPR region can be composed of a series of crRNAs. Each crRNA targeting a specific site is only a few dozen bases, and the entire carrier is small. Compared with ZFN and TALEN vectors, which are easier to construct (Research progress of CRISPR / Cas9 new gene targeting system. Journal of Jiangsu Agricultural Science. Li Hui et al., 2013). [0003] (CRISPR) / CRISPR-associated (Cas) is a continuously e...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/11C40B40/08
Inventor 李和刚赵金山张宝珣李培培刘华伟江科王建华侯乐乐杨培培孙友德
Owner 青岛市畜牧兽医研究所
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