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Cas9-mediated bombyx mori gene editing carrier and application

A gene editing, silkworm technology, applied in the field of genetic engineering, can solve the problems of gene loss of function, wrong transcription and translation, etc.

Active Publication Date: 2015-12-09
SOUTHWEST UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

And guide the Cas9 endonuclease to cut double-stranded DNA at the target site, and then the cell's non-homologous end joining repair mechanism (NHEJ) reconnects the genomic DNA at the break and introduces insertion or deletion mutations, causing target gene errors Transcription and translation of the gene, resulting in the loss of function of the gene

Method used

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  • Cas9-mediated bombyx mori gene editing carrier and application
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  • Cas9-mediated bombyx mori gene editing carrier and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1, Construction of Gene Editing Vectors Based on Cas9 and U6-sgRNA as Backbone

[0037] Log in to the addgene vector database (http: / / www.addgene.org / ), download the existing insect Cas9 gene and U6 sequences, and design the silkworm-specific Cas9 gene and U6-sgRNA sequences according to the codon characteristics of the silkworm. The sequences are SEQIDNo. 1 and SEQIDNo.2, and then sent to Gensript to synthesize the sequence.

[0038] The synthetic Cas9 gene sequence was digested by the ClaI and XbaI restriction sites and connected to the pSL1180 vector after the same digestion, and the ligation product was transformed into Escherichia coli E. coli DH5α competent cells, and then used LB containing ampicillin (Amp) Screen positive clones on the plate, pick a single colony of positive clones, and use it after enzyme digestion verification is correct; then connect the IE-1 promoter shown in SEQIDNo.3 at the XhoI and ClaI restriction sites, and connect the XbaI and ...

Embodiment 2

[0050] Example 2, Detection of CRISPR / Cas9 Edited Virus ie-1 Gene Inhibition of BmNPV Proliferation

[0051] Since the CRISPR / Cas9 gene editing system constructed in Example 1 can edit exogenous DNA in silkworm cells, it is assumed whether this system can be applied to silkworm anti-virus research. First, select the silkworm transcription initiation factor and replication key gene ie- 1 as the target gene for our gene editing. Similarly, predict the sgRNA sequence of the ie-1 gene according to the CRISPRdirect online analysis tool (http: / / crispr.dbcls.jp / ), and analyze the off-target efficiency of the target sequence in silkworm according to the software, and finally select the highly efficient sgRNA sequence , the sequence of which is shown in SEQ ID No.11-16, abbreviated as sgIE-1. According to the method of Example 1, the sgRNA sequence of the ie-1 gene was connected to the pIZ-U6-sgRNA expression vector digested by BbsI, and then transformed, and the positive clone was pi...

Embodiment 3

[0057] Example 3, Stable expression CRISPR / Cas9 system inhibits BmNPV proliferation effect detection

[0058] The insect cell expression vector pIZ-V5 / His developed by Invitrogen contains a Zeocin resistance gene, and a cell line stably expressing foreign proteins can be obtained through long-term antibiotic selection. In order to improve the antiviral effect of anti-gene editing vectors, the gene editing vectors pIZ-Cas9-Mcherry-U6-sgMock and pIZ-Cas9-Mcherry-U6-sgIE1 were transfected into BmN-SWU1 cells, and the transfection was stable for 48 hours Afterwards, positive cells were screened with a medium containing Zeocin antibiotic at a concentration of 400 μg / mL for about a week. After a large number of cells died, the concentration of antibiotics was gradually reduced, and finally cultured stably. The result is as Figure 11 As shown, basically more than 95% of the cells in about two months of screening contain the expression of the target protein, which proves that this c...

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Abstract

The invention discloses a Cas9-mediated bombyx mori gene editing carrier which comprises a constitutive gene editing carrier body and an inducible gene editing carrier body. The constitutive gene editing carrier body comprises an expression cassette U6-sgRNA with a U6 promoter regulating sgRNA expression, and an expression cassette with an IE-1 promoter regulating Cas9 expression. Expression of the inducible gene editing carrier body Cas9 is regulated by an Hr3-39k inducible promoter. A target gene can be stably and efficiently edited through the Cas9-mediated gene editing carrier, and a powerful tool is provided for preparing insect antiviral genetically modified materials and efficient antiviral varieties.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a Cas9-mediated gene editing carrier of silkworm, and also relates to the application of the gene editing carrier. Background technique [0002] CRISPR / Cas9 gene editing technology is one of the immune mechanisms that bacteria and archaea evolved under the long-term selection pressure of phages to effectively resist the invasion of foreign DNA. It is a piece of RNA that recognizes DNA through complementary base pairing, guides Cas9 endonuclease to cut the recognized double-stranded DNA, induces homologous recombination or non-homologous end joining, and then realizes editing on the target DNA. Because this new system has the advantages of simple preparation, low cost, and high efficiency compared with the previous gene editing system, it has achieved rapid development after development. Therefore, CRISPR / Cas9-based gene editing technology has great potential signif...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N5/10A01K67/04
Inventor 潘敏慧鲁成董战旗胡楠陈婷婷陈鹏
Owner SOUTHWEST UNIVERSITY
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