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Silkworm CRISPR/Cas12a mediated gene editing vector and application thereof

A gene editing, opie2-cas12a technology, applied in the field of genetic engineering, can solve the problems of Cas9 cannot be edited accurately, the protein is small, and the genome cannot be edited, so as to improve the anti-virus ability of silkworm

Inactive Publication Date: 2020-09-18
SOUTHWEST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, Cas9 technology also has some limitations. For example, the PAM sequence of Cas9 must be NGG. When the DNA fragment has no PAM, Cas9 cannot edit accurately; the recognition site is limited by the DNA sequence, and the entire genome cannot be edited, and there is a certain off-target efficiency.
With the discovery of the new CRISPR / Cas12a, whether it is the advantages of the Cas12a protein itself or the PAM required for cutting DNA, there are more choices. CRISPR / Cas12a can more comprehensively edit the sites on the genome, and its protein is smaller and simple in structure. , does not require tracrRNA; cutting can be far away from the recognition site, resulting in sticky ends after cutting; one promoter promotes multiple crRNAs to achieve multiple editing, and the off-target efficiency is lower

Method used

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  • Silkworm CRISPR/Cas12a mediated gene editing vector and application thereof
  • Silkworm CRISPR/Cas12a mediated gene editing vector and application thereof
  • Silkworm CRISPR/Cas12a mediated gene editing vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1, Construction of AsCas12a, FnCas12a and LbCas12a Gene Editing System Mediated by Bombyx mori CRISPR / Cas12a

[0026] In order to determine whether the CRISPR / Cas12a system can be used for gene editing in silkworms, the functions of three Cas12a enzymes (AsCas12a, FnCas12a and LbCas12a) used to edit mammalian cell genomes were tested, and the expression cassettes of AsCas12a, FnCas12a and LbCas12a were respectively constructed. Connect it with the nuclear localization signal and 3×HA tag, the expression frame is initiated by the OpIE2 promoter (SEQ ID NO.5) and terminated by the terminator OpIE2-PA. The specific steps are as follows: log in to the addgene vector database (http: / / www. addgene.org / ), download AsCas12a (SEQ ID NO.1), FnCas12a (SEQ ID NO.2) and LbCas12a (SEQ ID NO.3) sequence and U6 sequence (SEQ ID NO.4), wherein according to AsCas12a, FnCas12a and The PAM sequence of LbCas12a is selected by TTTG, and the PAM sequence of AsCas12a, FnCas12a and LbCa...

Embodiment 2

[0033] Example 2, analysis of the editing efficiency of the gene editing system of silkworm AsCas12a, FnCas12a and LbCas12a

[0034] In order to detect and analyze the editing of the BmNPV ie1 target gene by the three knockout systems AsCas12a, FnCas12a and LbCas12a, this study will knock out the vector Puro-OpIE2 prm -mCherry-OpIE2 prm -AsCas12a-U6-gie1, Puro-OpIE2 prm -mCherry-OpIE2 prm -FnCas12a-U6-gie1 and Puro-OpIE2 prm -mCherry-OpIE2 prm - After transfecting silkworm cells with LbCas12a-U6-gie1, the cell genome was extracted 48 hours after transfection, and the genome was used as a template for PCR amplification, and then the samples were sent for sequencing analysis. According to the position of the target site and the original sequence of the ie1 gene, the knockout of the target sequence is detected before and after the ie1 target site. From the analysis of the sequencing results, the three knockout systems AsCas12a, FnCas12a and LbCas12a all detected editing in t...

Embodiment 3

[0035] Example 3, analysis of the effect of the gene editing system of silkworm AsCas12a, FnCas12a and LbCas12a on inhibiting BmNPV

[0036] In order to further analyze whether the AsCas12a, FnCas12a and LbCas12a editing systems can well inhibit the proliferation and replication of the BmNPV genome after knocking out the BmNPV ie1 gene, this study respectively knocked out the vector Puro-OpIE2 prm -mCherry-OpIE2 prm -AsCas12a-U6-gIE1, Puro-OpIE2 prm -mCherry-OpIE2 prm -FnCas12a-U6-gIE1 and Puro-OpIE2 prm -mCherry-OpIE2 prm -LbCas12a-U6-gIE1 was transfected into silkworm cells for 48 hours, and after virus was added for 48 hours, the replication and proliferation of the virus gene gp41 in the control group and AsCas12a, FnCas12a and LbCas12a knockout groups were detected by fluorescence real-time quantitative PCR technology, and the control group and AsCas12a, FnCas12a were analyzed and LbCas12a knockout group's virus copy number difference. Analyze the expression of gp41 ...

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Abstract

The invention discloses a silkworm CRISPR / Cas12a mediated gene editing vector and an application thereof. The editing vector comprises an OpIE2-Cas12a expression cassette for regulating and controlling Cas12a expression by an OpIE2 promoter, wherein Cas12a can be AsCas12a, FnCas12a and LbCas12a; and a gene editing vector obtained by regulating and controlling gRNA expression through a U6 promoterhas a relatively good editing effect and antiviral capacity on BmNPV, the editing efficiency is high, a new direction can be provided for breeding of silkworm resistance materials, and a good technical platform is laid for gene function identification and research of organisms in the future.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a gene editing vector mediated by silkworm CRISPR / Cas12a, and also relates to the application of the editing vector. Background technique [0002] Clustered regularly interspaced short palindromic repeats (CRISPR) are widely used in the immune system of bacteria and archaea. As an editing tool, the CRISPR system can modify the genome at a fixed point, and its structure is divided into cas gene, leader sequence, crRNA (including repeat sequence and spacer sequence). At present, the CRISPR system has been widely used in drug development, disease treatment, animal models and biological genetic breeding. According to the number and sequence of Cas genes, the CRISPR / Cas system is divided into two types: Class 1 and Class 2. Since 2012, JINEK et al. found that Class2 Streptococcus pyogenes Cas9 (SpCas9), tracr-RNA and crRNA can target DNA in vitro after binding, and the Cas9 editin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/90C12N15/55A01K67/04
CPCA01K67/04A01K2217/075A01K2267/02C12N9/22C12N15/8509C12N15/902C12N2800/105
Inventor 潘敏慧董战旗张新铃秦琪陈鹏鲁成
Owner SOUTHWEST UNIV