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Cas9 system for efficiently editing bombyx mori genome and application of Cas9 system

A genome and silkworm technology, applied in the field of genes, can solve problems such as no baculoviruses, and achieve the effects of efficient editing, high transfection efficiency and good safety.

Pending Publication Date: 2021-05-28
SOUTHWEST UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Baculovirus obligately parasitizes arthropods, and is currently widely used in the expression of foreign proteins, and is also used in vaccine production, gene therapy, etc. There is no report on the use of baculovirus in the Cas9 system

Method used

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  • Cas9 system for efficiently editing bombyx mori genome and application of Cas9 system
  • Cas9 system for efficiently editing bombyx mori genome and application of Cas9 system
  • Cas9 system for efficiently editing bombyx mori genome and application of Cas9 system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Embodiment 1, establishment of Bombyx mori Cas9 tracer cell line

[0026] 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 SEQID No. .1 and SEQ ID No.2, and then sent to Gensript to synthesize the sequence.

[0027] The Cas9 gene sequence was cut by Hind III and Xba I on pIZ / V5-His, and the ligation product was transformed into Escherichia coli DH5α competent cells, and then positive clones were screened with LB plates containing bleomycin, and a single positive clone was picked. The colony was used after the enzyme digestion was verified correctly, and named pIZ-OpIE2-Cas9-pA. Ligate the Mcherry gene sequence (SEQ ID No.3) to the pIZ / V5-His vector at the Hind III and Kpn I restriction sites to obtain the pIZ-OpIE2-Mcherry-pA vector, which is used after verifi...

Embodiment 2

[0029] Embodiment 2, creation of Cas9 transgenic silkworm

[0030]First construct the pSL1180-IE1-Cas9-SV40 vector, digest the pSL1180-IE1-Cas9-SV40 and pBac-3Px3-EGFP vectors (SEQ ID No. 4) with AscI, and connect the excised IE1-Cas9-SV40 fragment to pBac -3Px3-EGFP vector, construct recombinant transgenic vector pBac-3Px3-EGFP-IE1-Cas9-SV40, IE1 promoter, 3Px3 promoter, EGFP gene sequences are SEQ ID No.5, SEQ ID No.6, SEQ ID No.7 shown.

[0031] By microinjection, the pBac-3Px3-EGFP-IE1-Cas9-SV40 vector was injected into the silkworm eggs (G0 generation) within 4 hours of laying, and randomly transposed into the silkworm genome under the action of piggyBac transposon, thereby constructing Transgenic silkworm. Since 3Px3 is an eye-specific promoter, the positive transgenic silkworms were screened by observing the EGFP fluorescence in the eyes of the G1 generation silkworms. The results are as follows image 3 , shown in A. The results showed that the expression of EGFP c...

Embodiment 3

[0039] Example 3. Construction of baculovirus packaging vector

[0040] U6-sgRNA (SEQ ID NO. 2) was digested by BamHI and EcoRI and inserted into pFastBac-Dual-HSP prm - EGFP-poly vector (referred to as pFBD vector) (this vector already contains polyhedrin, which is obtained by inserting polyhedrin promoter and polyhedrin gene into pFastBac-Dual vector through SphI and KpnI), the vector sequence is as SEQ ID No.20 As shown, the recombinant vector pFastBac-Dual-HSP was constructed prm -EGFP-poly-U6-sgRNA, referred to as pFBD-U6-sgRNA vector.

[0041] The viral gene lef11 and host genes HSPD1 and ATAD3A were selected as targets to design U6-sgRNA sequences respectively. The sgRNA sequences of lef11, HSPD1, and ATAD3A were predicted according to the CRI SPRdirect online analysis tool (http: / / crispr.dbcls.jp / ), and the off-target efficiency of the target sequences in Bombyx mori was analyzed according to the software, and finally the highly efficient sgRNA sequences were selecte...

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Abstract

The invention discloses a Cas9 system for efficiently editing a bombyx mori genome and an application of the Cas9 system. The system comprises a bombyx mori transgenic expression vector for expressing a Cas9 gene and bombyx mori nuclear polyhedrosis virus packaging virion containing an expression cassette U6-sgRNA expressed by a U6 promoter regulating sgRNA. A bombyx mori nuclear polyhedrosis virus (BmNPV) genome is used as a vector, the U6-sgRNA expression cassette is recombined to the virus genome through Bac-to-Bac transposition, a recombinant virion is constructed, a Cas9 cell line or Cas9 bombyx mori is infected with the recombinant virus, and a target gene can be effectively edited. The established Cas9 system does not need a transfection process, is high in transfection efficiency and good in safety, can stably and efficiently edit a target gene, and can be suitable for research on functions of viral genes and related host genes, a baculovirus expression system and the like.

Description

technical field [0001] The invention relates to the field of gene technology, in particular to a Cas9 system for efficiently editing the silkworm genome, and also relates to the application of the Cas9 system in editing the silkworm genome or the silkworm nuclear polyhedrosis virus gene. Background technique [0002] The CRISPR system is a powerful immune weapon evolved by bacteria and archaea in the process of resisting virus invasion. A guide RNA specifically recognizes the target DNA or RNA through base complementary pairing, and guides the effector protein with nuclease activity to cut the target. DNA or RNA, thereby removing foreign nucleic acids. The CRISPR / Cas9 system is widely used in basic research and applied research due to its single effector protein, simple operation, and high editing efficiency. The Chinese patent with the publication number CN105132460A discloses that the CRISPR / Cas9 system is used in the study of silkworm cell gene function and silkworm anti...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/866C12N9/22A01K67/04
CPCC12N15/8509C12N15/86C12N9/22A01K67/0339C12N2800/105C12N2710/14143A01K2227/706
Inventor 潘敏慧董战旗胡志刚陈鹏鲁成
Owner SOUTHWEST UNIVERSITY
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