CRISPR-Cas13d system for knocking down porcine epidemic diarrhea virus

A porcine epidemic diarrhea and virus technology, applied in the field of gene editing, can solve the problems of limited tools and achieve the effect of low off-target rate and high precision

Pending Publication Date: 2022-08-02
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, until recently, tools for disrupting or editing RNA molecules were limited

Method used

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  • CRISPR-Cas13d system for knocking down porcine epidemic diarrhea virus
  • CRISPR-Cas13d system for knocking down porcine epidemic diarrhea virus
  • CRISPR-Cas13d system for knocking down porcine epidemic diarrhea virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Example 1 Method for efficiently interfering with piglet epidemic diarrhea virus based on CRISPR-Cas13d system

[0066] 1. Construction of mCherry-PEDV-ORFs fluorescence reporter system

[0067] like figure 1 As shown, the ORF3, E, M and N sequences were amplified and fused to the C-terminus of the mCherry gene to form a fluorescent reporter system:

[0068] (1) mCherry-ORF3, figure 1 (A), SEQ ID NO: 1;

[0069] (2) mCherry-E, figure 1 (B), SEQ ID NO: 2;

[0070] (3) mCherry-M, figure 1 (C), SEQ ID NO: 3;

[0071] (4) mCherry-N, figure 1 (D), SEQ ID NO:4.

[0072] At the same time, a Cas13d expression vector mediated by nuclear signal NLS was constructed, and the synthetic NLS sequence was inserted into the head and tail of the Cas13d protein to obtain an engineered Cas13d expression vector NLS-Cas13d-NLS ( figure 2 , SEQ ID NO: 16).

[0073] Then proceed to the design of sgRNA. The Cas13d protease has no requirement for flanking sequences of the target and ...

Embodiment 2

[0170] Example 2 Method for efficient interference with piglet epidemic diarrhea virus based on CRISPR-Cas13d system

[0171] 1. Construction of mCherry-PEDV-ORFs fluorescence reporter system

[0172] like figure 1 As shown, the ORF3, E, M and N sequences were amplified and fused to the C-terminus of the mCherry gene to form a fluorescent reporter system:

[0173] (1) mCherry-PEDV-ORFs, figure 1 (A), SEQ ID NO: 10;

[0174] At the same time, construct the Cas13d expression vector mediated by the nuclear input signal NLS and the nuclear export signal NES, insert the synthetic NLS sequence or NES sequence into the head and tail of the Cas13d protein, and obtain the engineered Cas13d expression vector NLS-Cas13d-NLS, NES-Cas13d -NES( figure 2 , SEQ ID NOs: 16 and 17).

[0175] Then the design of tandem sgRNA was carried out. The Cas13d protease has no requirement for flanking sequences of the target and can target any RNA sequence.

[0176] Tandem sgRNAs targeting the sam...

Embodiment 3

[0244] Example 3 Knockdown of PEDV virus

[0245] Design engineered NLS- / NES-Cas13d for PEDV virus knockdown on vero cells.

[0246] (1) Vero cells were inoculated and cultured in DMEM high-glucose medium (Gbico) supplemented with 10% FBS, which contained penicillin (100 U / ml) and streptomycin (100 μg / ml).

[0247] (2) Divide into 24-well plates before transfection, and perform transfection when the density reaches 70%-80%.

[0248] (3) Transfection Take liposome transfection as an example. Follow Lipofectamine TM The operation manual of 3000Transfection Reagent (Invitrogen), mix 400ng NES- / NLS-Cas13d and 600ng pC0043-PspCas13b crRNAbackbone plasmid, co-transfect into each well of cells, change the medium after 6-8h (set different transfection groups, They are respectively used for the determination of viral titers in cell supernatants at different time periods, and the detection of viral protein expression in cells at specific time points).

[0249] (4) RNA virus infecti...

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Abstract

The invention provides a CRISPR-Cas13d (clustered regularly interspaced short palindromic repeats-associated protein 13d) system for knocking down a porcine epidemic diarrhea virus. An mCherry fluorescent reporter gene is utilized, four expression reading frame sequences of PEDV (porcine epidemic diarrhea virus) ORF3, E, M and N are fused to a C terminal of the mCherry gene respectively, a fluorescent reporter system for screening and targeting sgRNA efficient action targets of the PEDV expression reading frames is constructed, and the characteristic that CRISPR-Cas13d efficiently cuts mRNA is utilized to quickly screen efficient sgRNAs. Then, the screened sgRNAs which are combined in a high-efficiency targeting manner are utilized to carry out high-efficiency degradation on a target virus by using a CRISPR-Cas13d (clustered regularly interspaced short palindromic repeats) system. The RNA virus knock-down method provided by the invention has the advantages of high efficiency, high accuracy and low off-target rate.

Description

technical field [0001] The invention relates to the technical field of gene editing, in particular to a CRISPR-Cas13d system for knocking down porcine epidemic diarrhea virus. Background technique [0002] Virus is a non-cellular form composed of a nucleic acid molecule (DNA or RNA) and protein. Unlike the genetic material of general cellular organisms, which is double-stranded DNA, the genetic material of viruses can be DNA or RNA, and can be single-stranded or double-stranded. Judging from the viruses that have been discovered so far, more of them are RNA viruses. RNA viruses are a class of viruses that seriously endanger human health and agricultural safety production. Because of their rapid replication kinetics, high mutation rate and complex evolutionary dynamics, Prevention and treatment of related diseases have become particularly difficult. Each RNA virus outbreak causes a severe epidemic, and its fatality rate is high, resulting in huge economic losses. At presen...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/85C12N9/22
CPCC12N15/1131C12N15/85C12N9/22C12N2310/20C12N2320/10C12N2800/107Y02A50/30
Inventor 胡晓湘赵成成兰欣悦李果连迪李宁
Owner CHINA AGRI UNIV
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