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Transformed CRISPR/SaCas9 (clustered regularly interspaced short palindromic repeat/streptococcus pyogenes Cas9) system targeting at hepatitis B virus and application of system

A virus, enh2-pa1at technology, applied in the modified CRISPR/SaCas9 system targeting hepatitis B virus and its application field, which can solve the problems of single real-time quantitative PCR and so on

Active Publication Date: 2020-05-12
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, a large number of studies have proved that the CRISPR / Cas9 system can effectively inhibit the replication of HBV [1-6] , but before it can be put into therapeutic application, several important issues need to be solved: first, how to efficiently and safely deliver the CRISPR / Cas9 system; At the same time as the target site, it is also possible to cut DNA sequences similar to the target site
Anti-HBV replication of previously published AAV-delivered CRISPR-SaCas9 system [7,8] Both have limitations: First, in the above two studies, the detection of virus indicators is limited, only some virus antigens are the main ones, and Yu Liu et al. A single method; secondly, different serotypes of AAV have different tissue tropisms. Although the above studies all use AAV8 serotype as the carrier with better liver tissue-specific delivery, no further research has been done to reduce the possibility of off-target effects. Improve

Method used

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  • Transformed CRISPR/SaCas9 (clustered regularly interspaced short palindromic repeat/streptococcus pyogenes Cas9) system targeting at hepatitis B virus and application of system
  • Transformed CRISPR/SaCas9 (clustered regularly interspaced short palindromic repeat/streptococcus pyogenes Cas9) system targeting at hepatitis B virus and application of system
  • Transformed CRISPR/SaCas9 (clustered regularly interspaced short palindromic repeat/streptococcus pyogenes Cas9) system targeting at hepatitis B virus and application of system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] [Example 1] Design, synthesis and eukaryotic expression vector construction of gRNA sequences targeting 22 different genotypes of HBV conservative sequences

[0060] 1. Selection and design of gRNA targeting HBV DNA

[0061] Using the D-type HBV (Genebank ID: V01460.1) as the reference sequence, the target site with a higher score on the HBV DNA was found through the Crisprdesign tool on crispr.mit.edu, and the target site sequence was in the form of 5'-(20N )-NNGRRT3' or 5'-ARRCNN(20N)-3'. Different from previous designs, the present invention compares the HBV DNA sequences of 22 different genotypes, and selects the gRNA sequences located in highly conserved regions from the found target sites. These conserved regions themselves are also genes in the replication process. Regions with low mutation rates. 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 gRNA sequences and targ...

Embodiment 2

[0078] [Example 2] Verifying the inhibition of HBV replication by the gRNA-guided CRISPR / Cas9 system of the present invention at the cellular level

[0079] In order to verify whether the gRNA-guided CRISPR / SaCas9 system designed in the present invention has the ability to resist HBV replication, the clone of the constructed gRNA will be combined with the replicon prcccDNA (genotype D; Genebank ID: V01460. 1), pCMV-Cre was co-transfected into the liver cancer cell line Huh7, and another gRNA targeting GFP (T GFP ) were 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 48 hours of transfection, it was observed that the growth state of the Huh7 cell line was good, and 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 ...

Embodiment 3

[0161] [Example 3] Differential expression levels of different candidate promoters in liver-derived and non-liver-derived cell lines

[0162] 1. According to the genome structure of D-type HBV (Genebank ID: V01460.1), select the corresponding four promoters Enh1 / X, Enh2 / C, preS1, preS2, and one enhancer Enh2 as candidates; according to human and rat genomes Information selected three promoters PPck (Gene ID: 362282), Palb (Gene ID: 111832673), Pa1AT (Gene ID: 5265) with liver-specific expression, and one enhancer Ealb as candidates. Finally, 13 promoter combinations were selected: Enh1 / X, Enh2 / C, preS1, preS2, PPck, Ealb-PPck, Enh2-PPck, Palb, Ealb-Palb, Enh2-Palb, Pa1AT, Ealb-Pa1AT, Enh2-Pa1AT.

[0163] 2. Ligate the above promoter fragment and CMV promoter with the pGL3 luciferase reporter plasmid treated with Sac1 enzyme and Hind3 enzyme;

[0164] 3. The 14 plasmids constructed in the above 2 were co-transfected with the plasmid phRL-TK carrying the Renilla luciferase gene...

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Abstract

The invention discloses a transformed CRISPR / SaCas9 (clustered regularly interspaced short palindromic repeat / streptococcus pyogenes Cas9) system targeting at hepatitis B virus and application of thesystem. According to designing principles of gRNA (guide ribonucleic acid) of CRISPR and conservative regions of HBV (hepatitis B virus) sequences of different genotypes, three gRNAs are screened andconstructed on a PX601expression vector, meanwhile, different specific liver promoters are selected and combined with an enhancer of an HBV genome, and two promoter combinations are screened to transform the expression vector PX601. When the rRNA guided CRISPR transformation system is applied to cell models and mouse models, expression and replication of hepatitis B virus genes can be effectivelyinhibited while liver tissue expression specificity of SaCas9 is remarkably improved. The system is easy to operate, high in security and high in HBV replication. The transformed CRISPR / SaCas9 systemhas the potential of being a novel treatment medicine for treating hepatitis B virus.

Description

technical field [0001] The invention belongs to the technical field of gene mutation and genetic engineering, and specifically relates to the transformation of the CRISPR / SaCas9 nuclease system that can be delivered by an adeno-associated virus (AAV) vector, and the selection of efficient and safer liver-specific expression of SaCas9 nuclease Promoter combinations, and design, construct, and screen the optimal guide RNA and its target site sequence for the conserved sequences of various genotypes and subtypes of hepatitis B virus. Background technique [0002] Hepatitis B virus (HBV) is a virus that can cause chronic infection in patients and increase the risk of liver cirrhosis and liver cancer. According to statistics, about 291 million people in the world are infected with hepatitis B virus, and about 1 million people lose their lives every year because of hepatitis B and the liver cirrhosis and liver cancer it causes. Therefore, viral hepatitis B is an important problem...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/864C12N15/90A61K48/00A61P1/16A61P31/20
CPCC12N9/22C12N15/113C12N15/86C12N15/907A61K48/0058A61K48/0008A61K9/0019A61P1/16A61P31/20C12N2310/20C12N2750/14143
Inventor 陈宇严鲲冯姜澎刘杏
Owner WUHAN UNIV
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