Screening method of sgRNA (small guide ribonucleic acid) efficient action target based on CRISPR-Cas13d (clustered red regularly interspaced short palindromic repeat) system and application

A screening method, RNA virus technology, applied in the field of gene editing, can solve problems such as unsatisfactory effects, complex life activities, and difficult treatment of RNA virus-related diseases, and achieve low off-target rate, high precision rate, and high accuracy.

Active Publication Date: 2020-01-07
CHINA AGRI UNIV
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Problems solved by technology

Due to its characteristics of high variability, rapid replication, and complex life activities, the prevention and control of RNA viruses and the treatment of related diseases have become particularly difficult
The main prevention and treatment ...

Method used

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  • Screening method of sgRNA (small guide ribonucleic acid) efficient action target based on CRISPR-Cas13d (clustered red regularly interspaced short palindromic repeat) system and application
  • Screening method of sgRNA (small guide ribonucleic acid) efficient action target based on CRISPR-Cas13d (clustered red regularly interspaced short palindromic repeat) system and application
  • Screening method of sgRNA (small guide ribonucleic acid) efficient action target based on CRISPR-Cas13d (clustered red regularly interspaced short palindromic repeat) system and application

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Embodiment 1

[0046] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the examples are all in accordance with conventional experimental conditions, such as Sambrook et al. Molecular Cloning Experiment Manual (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual, 2001), or in accordance with the conditions suggested by the manufacturer's instructions. Example 1 Screening of sgRNA high-efficiency targets for PRRSV based on CRISPR-Cas13d system

[0047] 1. Construction of mCherry-ORF4 / ORF5 fluorescent reporter system

[0048] Such as figure 1 As shown, after the ORF4 / ORF5 sequence is amplified, it is fused to the carbon-terminus (C-terminus) of the mCherry gene to form the following two fluorescent reporter systems:

[0049] (1) mCherry-ORF4, figure 1 (A), SEQ ID NO: 1;

[0050] (2) mCherry-ORF5, figure 1 (B), SEQ ID NO: 2;

[0051] The construction method of the r...

Embodiment 2

[0089] The results showed that the mCherry-ORF4 / 5 reporter plasmid mRNA was knocked down efficiently, with an efficiency of up to 96%, among which ORF4-sgRNA1 (SEQ ID NO:5) and ORF5-sgRNA1 (SEQ ID NO:6) had the best effects, The biological reproducibility is the best, and it is selected as the sgRNA knocked down by the subsequent PRRSV-GFP recombinant virus ( image 3 , A and B). The knockdown of embodiment 2PRRSV-GFP recombinant virus

[0090] Engineered NLS- / NES-Cas13d was designed to knock down PRRSV-GFP recombinant virus on Marc145 cells.

[0091] (1) Marc145 cells were inoculated and cultured in DMEM high-glucose medium (HyClone, SH30022.01B) supplemented with 10% FBS, which contained penicillin (100 U / ml) and streptomycin (100 μg / ml).

[0092] (2) Divide into 24-well plates before transfection, and perform transfection when the density reaches 70%-80%.

[0093] (3) Transfection Take liposome transfection as an example. Follow Lipofectamine TM 2000 Transfection Reage...

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Abstract

The invention provides a screening method of a sgRNA (small guide ribonucleic acid) efficient action target based on a CRISPR-Cas13d (clustered red regularly interspaced short palindromic repeat) system and application, and particularly application in RNA virus knockdown. According to the screening method, two expression read frame sequences ORF4 and ORF5 of a PRRSV are respectively fused with a carbon end of a mCherry gene through a mCherry fluorescence report gene, a fluorescence report system for screening sgRNA efficient action targets of the CRISPR-Cas13d system is established, and by virtue of the property that mRNA is efficiently cut by CRISPR-Cas13d, rapid screening of sgRNAs with high efficiency is implemented. Additionally, PRRSV-GFP recombinant viruses are efficiently degraded by using the screened efficient targetedly combined sgRNA based on the CRISPR-Cas13d system. The RNA virus knockdown method provided by the invention has the advantages of being high in efficiency, high in precision and low in target missing rate.

Description

technical field [0001] The invention relates to the technical field of gene editing, in particular to a screening method and application of sgRNA high-efficiency targets based on the CRISPR-Cas13d system. Background technique [0002] RNA viruses are a class of viruses that seriously endanger human health and agricultural production safety. Due to its characteristics of high variability, rapid replication, and complex life activities, the prevention and control of RNA viruses and the treatment of related diseases have become particularly difficult. The main prevention and treatment methods for common RNA viruses and related diseases are the research and development of vaccines, antibodies, and RNA interference technology (RNAi), but the results are not very satisfactory, and the exploration and development of new methods is imminent. [0003] In recent years, gene editing technology mediated by programmable nucleases has developed rapidly. CRISPR (Clustered regularly inters...

Claims

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Application Information

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IPC IPC(8): C12N15/79C12N15/113
CPCC12N15/113C12N15/79C12N2310/20
Inventor 胡晓湘李果王鑫杰李宁
Owner CHINA AGRI UNIV
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