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GRNA for targeted destruction of SARS-CoV-2 genome and application of gRNA

A virus genome, sars-cov-2 technology, applied in the field of gene editing

Active Publication Date: 2020-12-29
GUANGZHOU REFORGENE MEDICINE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In September 2019, some researchers used this system to successfully achieve targeted cleavage of three RNA viruses (lymphocytic virus LCMV, influenza A virus IAV, and vesicular stomatitis virus VSV) in cultured cells in vitro. It is as effective as shRNA-mediated RNA interference
[0005] No viable Cas13d gene-editing therapy for SARS-CoV-2 viral infection has yet been invented

Method used

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  • GRNA for targeted destruction of SARS-CoV-2 genome and application of gRNA
  • GRNA for targeted destruction of SARS-CoV-2 genome and application of gRNA
  • GRNA for targeted destruction of SARS-CoV-2 genome and application of gRNA

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Reporter carrier design and construction.

[0055] The psiCHECK2 plasmid was linearized with XhoI and NotI double restriction enzymes, and reacted at 37°C for 1 hour. The digested product was subjected to 1% agarose gel electrophoresis, and the 6242bp digested product was recovered by cutting the gel, and stored at -20°C for future use.

[0056] According to the published SARS-CoV-2 genome sequence (NC_045512.2), its S region was synthesized into the pcDNA6B vector. SARS-CoV-2 genome structure such as figure 1 shown.

[0057] Use PrimeSTAR Max DNA polymerase premix to perform PCR amplification of bases 24-3804bp in the Spike region, and at the same time, a 15-20bp homology arm is introduced into the primers for subsequent cloning between the NotI and XhoI restriction sites of psiCHECK2 , as the 3'UTR of hRluc. The PCR products were subjected to 1% agarose gel electrophoresis, and the target bands were recovered by cutting the gel.

[0058] The PCR recovery product ...

Embodiment 2

[0063] gRNA vector design and construction.

[0064] A 30nt gRNA targeting domain sequence was designed according to the target sequence in the S region of the SARS-CoV-2 genome, and two gRNAs were designed for the hRluc region as a control. The targeting domains of the gRNA are shown in Table 1.

[0065] Table 1. Targeting domains of gRNAs

[0066] name targeting domain serial number nCoV-S-1 AUAACCCACAUAAUAAGCUGCAGCACCAGC SEQ ID NO:1 nCoV-S-2 UUAGAAUUCCAAGCUAUAACGCAGCCUGUA SEQ ID NO:2 nCoV-S-3 UAGAACCUGUAGAAUAAACACGCCAAGUAG SEQ ID NO:3 nCoV-S-4 UCACCAUAUUUGUUUGAUGAAGCCAGCAUCU SEQ ID NO:4 nCoV-S-5 ACUCUGACAUUUUAGUAGCAGCAGCAAGAUUAG SEQ ID NO:5 nCoV-S-6 AGCAGGAUCCACAAGAACAACAGCCCUUGA SEQ ID NO:6 nCoV-S-7 CAGAGACAUGUAUAGCAUGGAACCAAGUAA SEQ ID NO:7 hRluc-1 ACAAAGAUGAUUUUCUUUGGAAGGUUCAGC SEQ ID NO:8 hRluc-2 CGAAGAAGUUAUUCUCAAGCACCAUUUUCU SEQ ID NO:9

[0067] Synthesize Oligo DNA corr...

Embodiment 3

[0076] Plasmid transfection and dual luciferase reporter gene detection.

[0077] 293T cells were inoculated into a 24-well plate in a certain amount so that the confluence of the cells reached about 80% after 24 hours.

[0078] Using Lipofectamine 2000 liposome transfection reagent, the pXR004 plasmid with gRNA cloned and the pXR001 plasmid expressing Cas13d (the vector map of pXR001 is shown in Figure 4shown), psiCHECK2-nCoV-S plasmid was co-transfected with the above 293T cells at a ratio of 1:1:1, the psiCHECK2-nCoV-S plasmid was transfected alone as the negative control group, and the cells not transfected with any plasmid were used as the blank control . Three replicate wells (A / B / C) were set up for each transfection. The plasmid transfection groups are shown in Table 2.

[0079] Table 2. Plasmid transfection groups

[0080]

[0081]

[0082] 48 hours after the transfection of the plasmid, according to the method of using the dual-luciferase reporter gene dete...

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PUM

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Abstract

The invention relates to gRNA for targeted destruction of an SARS-CoV-2 genome and an application of the gRNA, and belongs to the technical field of gene editing. The gRNA comprises a targeting structural domain, and a sequence of the targeting structural domain targets an S region of the SARS-CoV-2 genome. The invention also provides a gRNA expression vector for treating SARS-CoV-2 infection, a Crispr system, a composition containing the gRNA or nucleotide encoding the gRNA, and a pharmaceutical application of the gRNA expression vector, the Crispr system and the composition. The gRNA provided by the invention can destroy the Spike region of the SARS-CoV-2 genome in a targeting manner, and can be used for treating the SARS-CoV-2 infection.

Description

technical field [0001] The invention relates to the technical field of gene editing, in particular to a gRNA targeted to destroy the genome of SARS-CoV-2 virus and its application. Background technique [0002] There are many types of coronaviruses, most of which only infect animals but sometimes mutate and infect humans. The novel coronavirus, SARS-CoV-2, is a mutated virus that can spread rapidly from person to person and can cause severe respiratory disease. It is highly contagious and harmful, and there is currently no specific drug or method that can treat the new coronavirus. Although some researchers have proposed that some antiviral drugs on the market or under research may have an inhibitory effect on the new coronavirus, their effectiveness and safety have not been proven, and further clinical research is needed. Therefore, the development of new therapeutic techniques and drugs has become an urgent need. [0003] SARS-CoV-2 belongs to coronavirus lineage B (Bet...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N9/22C12N15/63A61K48/00A61K47/69A61K47/59A61P31/14
CPCC12N15/113C12N9/22C12N15/63A61K48/0025A61K48/005A61K47/6929A61K47/59A61P31/14C12N2310/20
Inventor 梁峻彬欧家裕皇甫德胜徐辉古博
Owner GUANGZHOU REFORGENE MEDICINE CO LTD
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