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sgRNAs that efficiently edit the porcine miR-17-92 gene cluster

A gene clustering and editing technology, applied in DNA/RNA fragments, genetic engineering, DNA preparation, etc., can solve problems such as limiting the breeding and application prospects of transgenic pigs, and achieve the effect of removing potential biological safety hazards and increasing safety.

Active Publication Date: 2021-12-03
广东明珠生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These factors limit the breeding and application prospects of transgenic pigs

Method used

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  • sgRNAs that efficiently edit the porcine miR-17-92 gene cluster
  • sgRNAs that efficiently edit the porcine miR-17-92 gene cluster
  • sgRNAs that efficiently edit the porcine miR-17-92 gene cluster

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] 1-1. Design of sgRNA sequence and construction of PX330 expression vector

[0026] Three sgRNA sequences targeting porcine miR-17-92 were designed and synthesized. The sgRNA sequences designed above were synthesized; the DNA sequences of the six single-stranded sgRNAs were annealed to form three oligonucleotide chains of sgRNAs targeting different positions of the porcine miR-17-92 3'-UTR; This oligonucleotide was then ligated into the pX330 plasmid vector.

[0027] The sequences of the three sgRNAs and the sequences of their action sites are:

[0028] SgRNA-1 sequence: 5-atgattctgtaccacttgtg-3

[0029] The sequence of the sgRNA-1 action site: 5-CACAAGTGGTACAGAATCAT-3

[0030] SgRNA-2 sequence: 5-GCTGTATTGTCAGATTTATC-3

[0031] The sequence of the sgRNA-2 action site: 5-GATAAATCTGACAATACAGC-3

[0032] SgRNA-3 sequence: 5-attctgtaccacttgtgagg-3

[0033] The sequence of the sgRNA-3 action site: 5-CCTCACAAGTGGTACAGAAT-3

[0034] Among them, the present invention inv...

Embodiment 2

[0043] Construction of targeting vector for site-directed integration of shRNA

[0044] According to the screened high-efficiency sgRNA, the shRNA site-specific integration targeting vector (pLB-shRNA-KI-Donor) matching the sgRNA was designed and constructed. The main components of the targeting vector are: upstream homology arm, shRNA-EGFP gene, downstream homology Source arm and backbone vector for prokaryotic expression. The shRNA site-specific integration targeting plasmid and the screened sgRNA can specifically genetically modify the porcine miR-17-92 site, and then combine the method of fluorescence microscopy and PCR to easily analyze the recognition of foreign genes by the sgRNA Feasibility of site integration and expression ( figure 2 , schematic diagram of shRNA site-specific targeting plasmid vector).

Embodiment 3

[0046] Co-transfection of PX330 plasmid and pLB-shRNA-KI-Donor plasmid

[0047] Resuscitate PK-15-EGFP-KI cells, and when they are close to confluence, wash 2~3 times with DPBS, discard the supernatant, add electrotransfection buffer, and then press PX330 plasmid and pLB-shRNA-KI-Donor plasmid Add the ratio to the cells and the buffer solution, mix gently with a pipette, gently transfer the mixture into the electrode cup, and place the electroporation cup on the electroporation instrument for electric shock operation. After the electric shock was completed, the electro-rotator cup was left to stand for 10 minutes, and then the mixed solution in the electro-rotor cup was transferred to a cell culture dish. Finally, the cell culture dish was placed in a 37°C carbon dioxide incubator for cultivation. After 12 hours of incubation, the medium was changed.

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Abstract

The present invention provides an sgRNA capable of effectively editing the porcine miR-17-92 gene cluster. On the premise that the sgRNA can specifically recognize the porcine miR-17-92 gene cluster, the shRNA is successfully integrated into the porcine site using CRISPR / Cas9 and RNAi technology. Kidney cell PK‑15‑EGFP‑KI cells were screened for positive cell clones that knocked down the expression of the EGFP gene; the above sgRNA was used to site-specifically integrate shRNA into pig fetal fibroblasts, and a positive cell line for site-specific integration of shRNA was successfully screened out. The stable transcription and site-specific integration events of shRNA were detected. During the preparation of the cell line, the shRNA was stably transcribed and effectively expressed under the action of the promoter of the porcine miR‑17‑92 gene cluster without introducing any foreign promoter Subgenes and positive and negative screening marker genes increase the safety of transgenic pigs and are of great significance for removing the biological safety hazards of transgenic pigs.

Description

technical field [0001] The invention discloses an sgRNA capable of effectively editing the porcine miR-17-92 gene cluster and its application, and also discloses a site-specific strategy and feasibility analysis of a safer exogenous gene related to the sgRNA, belonging to the field of biotechnology. [0002] technical background [0003] CRISPR / Cas9 was first discovered in bacteria and archaea, and is an adaptive immune defense mechanism evolved by organisms in response to constant attacks by viruses and plasmids. The working principle of the CRISPR / Cas9 system is that crRNA (CRISPR-derived RNA) combines with tracrRNA (trans-activating RNA) through base pairing to form a tracrRNA / crRNA complex, which guides the nuclease Cas9 protein to sequence targets paired with crRNA Site cuts double-stranded DNA. By artificially designing these two RNAs, a sgRNA (single-guide RNA) with a guiding effect can be engineered, which is sufficient to guide the site-specific cutting of DNA by Ca...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/10C12N15/85C12N5/10A01K67/027
CPCA01K67/0275A01K2227/108C12N15/113C12N15/85C12N2310/10C12N2800/80C12N2810/10
Inventor 逄大欣陆超欧阳红生
Owner 广东明珠生物技术有限公司
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