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Method for specifically knocking out hepatitis B virus by CRISPR/Cas9 and gRNA applied to specific targeting HBV DNA

A hepatitis B virus, specific technology, applied in the field of genetic engineering, can solve problems such as unsatisfactory suppression effect, high HBV gene mutation rate, and low HBV gene mutation rate

Active Publication Date: 2015-04-08
浙江安维珞诊断技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Previously published studies based on the CRISPR / Cas9 system against HBV replication have limitations (Su-Ru Lin, et al., Molecular Therapy-Nucleic Acids, 2014, 3, e186; Christoph Seeger and Ji A Sohn, Molecular Therapy-Nucleic Acids Acids, 2014, 3, e216): First of all, gRNAs were designed and experimented with a single genotype HBV sequence as a template, and the conserved region of the HBV sequence (that is, the region with a low HBV gene mutation rate) was not selected
Due to the high gene mutation rate of HBV virus, the gRNA designed for a single genotype of HBV has a high off-target probability, and cannot inhibit the replication of other genotypes of HBV, so its later clinical transformation may have greater limitations; secondly , Christoph Seeger only detected the expression level of some HBV antigens at the cellular level. Although the mouse model was verified in the report of Su-Ru Lin, the inhibitory effect was not satisfactory (only about 20%)

Method used

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  • Method for specifically knocking out hepatitis B virus by CRISPR/Cas9 and gRNA applied to specific targeting HBV DNA
  • Method for specifically knocking out hepatitis B virus by CRISPR/Cas9 and gRNA applied to specific targeting HBV DNA
  • Method for specifically knocking out hepatitis B virus by CRISPR/Cas9 and gRNA applied to specific targeting HBV DNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] [Example 1] Design, synthesis and eukaryotic expression vector construction of gRNA sequences targeting 26 different genotypes of HBV conservative sequences

[0048] 1. Selection and design of gRNA targeting HBV DNA

[0049] Using the D-type HBV (Genebank ID: V01460.1) as the reference sequence, use the Crispr design tool on crispr.mit.edu to find the target site with a higher score on the HBV DNA. The target site sequence is in the form of 5'-N (20)-NGG3' or 5'-CCN-N(20)-3'. Different from the previous design, we compared the HBV DNA sequences of 26 different genotypes, and selected gRNA sequences located in highly conserved regions from the found target sites. These conserved regions themselves are also gene mutations during the replication process. low rate areas. At the same time, the human genome sequence was compared, gRNAs with high homology to the human genome sequence were excluded, and a series of brand new, unreported gRNA sequences and targets were fin...

Embodiment 2

[0075] [Example 2] Verifying the inhibition of HBV replication by the gRNA-guided CRISPR / Cas9 system of the present invention at the cellular level

[0076] In order to verify whether the gRNA-guided CRISPR / Cas9 system designed in the present invention has the ability to resist HBV replication, the clone of the gRNA constructed in the present invention was combined with the replicon pHBV1.3 of hepatitis B virus (genotype D; Genebank ID: V01460.1) were co-transfected into the liver cancer cell line HepG2, and another gRNA targeting GFP (T GFP ) (SEQ ID NO. 18) was used as a control group. To normalize the transfection efficiency between different wells, each well was transfected with the same amount of β-galactosidase expression plasmid pSVβ-gal. After 24, 48, and 72 hours of transfection, the culture supernatant was taken to measure the expression levels of hepatitis B virus surface antigen (HBsAg) and hepatitis B virus e antigen (HBeAg). The results showed that 8 diffe...

Embodiment 3

[0131] [Example 3] Inhibition of HBV replication by the combined use of any two or more gRNAs

[0132] When we use multiple gRNAs in combination, the CRISPR / Cas9 system can cut two or more sites on HBV DNA at the same time, so that two or more sites can be mutated at the same time, thereby improving the inhibition efficiency of HBV. Especially when Tmix is ​​used in combination, multiple HBV DNA breakpoints may be generated due to untimely repair, which will further degrade HBV DNA, reduce the survival rate of HBV, and greatly improve the efficiency of the CRISPR / Cas9 system to clear HBV. In the present invention, HepG2 cells were co-transfected with Tmix, pHBV1.3, and pSVβ-gal, a combination of 8 gRNAs. After 2 or 4 days of culture, ELISA, Northern blot, and qPCR were used to detect their inhibitory efficiency against HBV. From the experimental results, it can be seen that the inhibitory effect of Tmix on hepatitis B virus is significantly better than that of any gRNA alo...

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Abstract

The invention relates to the technical field of molecular biology and biological medicines, and particularly relates to application of gRNA sequences and combination thereof based on a CRISPR system in treatment on hepatitis B virus. According to the method disclosed by the invention, eight types of guidance RNAs (gRNA) are designed according to design rules of CRISPR gRNA and a conservative region of different genotypes of HBV sequences, and the eight types of guidance RNAs are structured on a PX330 expression vector. By utilizing the eight gRNAs in a cell model, a mouse model and a CRISPR / Cas9 system guided by combination of the cell model and the mouse model, the expression and replication of the hepatitis B virus can be effectively inhibited. By united application of a plurality of gRNAs, a better effect can be achieved, and different genotypes of HBV replication can be better inhibited. The system has the characteristics of being easy to operate, high in inhibition efficiency on HBV replication and applicable to various genotypes. Therefore, the gRNA and the combination thereof related to the invention are expected to be applied in preparation of a novel drug for treating the hepatitis B virus.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and more specifically relates to a method for specifically knocking out hepatitis B virus by CRISPR / Cas9 and a gRNA for specifically targeting HBV DNA. Background technique [0002] Hepatitis B virus (HBV) is a virus that can cause a variety of liver diseases, including hepatitis, liver fibrosis, and hepatocellular carcinoma. Worldwide, there are nearly 350 million people with chronic hepatitis B infection, and nearly one-third of them are in my country. Hepatitis B virus has brought great challenges to the medical and health system of the whole world, especially our country. Currently, there are two main treatment methods for HBV: 1. Alpha interferon (IFN-α) and pegylated alpha interferon (PEG-IFN-α). It is believed that it may be through direct inhibition of virus replication or immune regulation; 2. Nucleoside analogs, the current clinical use of nucleoside analogs mainly includes lami...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/85A61K48/00A61P31/20
Inventor 陈宇郭德银郝瑞栋刘杏
Owner 浙江安维珞诊断技术有限公司
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