Effective Cas13a-based anti-dengue virus nucleic acid target and application thereof

A dengue virus and target technology, which is applied in the field of nucleic acid targets against dengue virus, can solve the problems of inability to target and clear RNA viruses, and achieve the effects of less drug resistance, strong specificity, and high efficiency

Active Publication Date: 2018-10-30
ACADEMY OF MILITARY MEDICAL SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the field of anti-virus, researchers have successfully used CRISPR-Cas9 technology to achieve precise targeting and high-efficiency inhibition of DNA viruses such as HPV and HBV. However, the CRISPR-Cas9 system can only achieve DNA editing and DNA virus targeting. Clearance, it is not yet possible to directly target and clear RNA viruses

Method used

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  • Effective Cas13a-based anti-dengue virus nucleic acid target and application thereof
  • Effective Cas13a-based anti-dengue virus nucleic acid target and application thereof
  • Effective Cas13a-based anti-dengue virus nucleic acid target and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1. Preparation and screening of CRISPR-Cas13a target sequence targeting dengue virus

[0037] 1. Design and synthesis of crRNA sequence targeting dengue virus DENV-2

[0038] Taking the genome sequence (Genebank ID: U87411) of type 2 dengue virus DENV-2 (strain 16681) as the reference sequence, for the 6 genes (PrM, NS2a, NS2b, NS3, NS4a, 3'UTR) of type 2 dengue virus, A continuous 29 nucleotide sequence was selected in each gene sequence as the target sequence of the CRISPR-Cas13a system.

[0039] The selected 6 target sequences were respectively reverse-complemented to obtain 6 reverse-complementary strands;

[0040] Then add GGGGATTTAGACTACCCCAAAAACGAAGGGGACTAAAAC to the 5' end of the sequence of the 6 reverse complementary strands to synthesize the forward oligonucleotide sequence (Table 1);

[0041] Using the forward oligonucleotide sequence as a template, use T7-crRNA-F: TAATACGACTCACTATAGGGGATTTAGACTACCCCAA as an upstream primer, and use R-NNN (Table 1:...

Embodiment 2

[0076] Example 2. Efficiency of CRISPR-Cas13a system targeting crRNA-NS3 inhibiting dengue virus replication and expression in vitro

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Abstract

The invention discloses an effective Cas13a-based anti-dengue virus nucleic acid target and application thereof. The invention provides a CRISPR-Cas13a system for inhibiting dengue virus, wherein theCRISPR-Cas13a system comprises a Cas13a protein and a crRNA corresponding to an NS3 gene target of the dengue virus, or a complex formed by the Cas13a protein and crRNA; and NS3 gene targets of the dengue virus are 4657th-4685th nucleotide sequences of the NS3 gene. In the invention, a novel anti-dengue virus method is found, is different from a traditional anti-virus method, directly targets a viral nucleic acid, specifically degrades a target gene, and makes the virus lose the replication ability; therefore, the effective Cas13a-based anti-dengue virus nucleic acid target has characteristicsof high efficiency, high specificity and programmability, is not easy to produce drug resistance generated by traditional antiviral drugs, and may become a novel antiviral drug in the future.

Description

technical field [0001] The invention belongs to the technical field of gene mutation and genetic engineering, and relates to an effective Cas13a-based anti-dengue virus nucleic acid target and application thereof. Background technique [0002] With global warming and increasing population mobility, dengue fever is increasingly becoming an important infectious disease in tropical and subtropical regions. Dengue virus is the pathogen of dengue fever and is divided into four serotypes, named DENV-1 and DENV-2 respectively. , DENV-3 and DENV-4. About 50 million to 100 million people are infected with dengue virus every year, and about 1% of the infected people develop into severe patients who need hospitalization, manifest as dengue hemorrhagic fever and dengue shock syndrome, and even die. The dependent virus infection enhancement (antibody-dependent enhancement, ADE) has led to difficulties in the development of dengue virus vaccines, and there is no specific therapeutic drug...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N9/22A61K31/7088A61K39/12A61P31/14
CPCA61K31/7088A61K39/12A61P31/14C12N9/22C12N15/1131C12N2310/10C12N2770/24134Y02A50/30
Inventor 寇志华李浩宋宏彬周育森李军锋王珊董雪郭彦谢靖贾雷立刘鸿博邱少富杨超杰
Owner ACADEMY OF MILITARY MEDICAL SCI
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