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CRISPR-Cas9 targeted knockout hepatitis b virus cccDNA and specific sgRNA thereof

A hepatitis B virus, specific technology, applied in the field of genetic engineering, can solve the problems of low efficiency and reduction

Active Publication Date: 2014-07-09
AOMIAO BIOTECH GUANGZHOU CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Aiming at the existing problems of targeting HBV cccDNA: (1) low efficiency, only a small amount of HBV cccDNA can be knocked out; (2) can only slightly reduce the expression of HBsAg, wait

Method used

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  • CRISPR-Cas9 targeted knockout hepatitis b virus cccDNA and specific sgRNA thereof
  • CRISPR-Cas9 targeted knockout hepatitis b virus cccDNA and specific sgRNA thereof
  • CRISPR-Cas9 targeted knockout hepatitis b virus cccDNA and specific sgRNA thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0092] Example 1 Design and synthesis of sgRNA for specifically targeting cccDNA in CRISPR-Cas9 specific knockout of hepatitis B virus cccDNA

[0093] Because in vitro transcription is not used, it is only made by constructing a common vector. Therefore, unless otherwise specified, the sgRNA sequence in the text refers to the DNA sequence corresponding to the sgRNA.

[0094] 1. Design and selection of sgRNA targeting HBV cccDNA

[0095] (1) Select the 5'-GGN(19)GG sequence on the HBV cccDNA. If there is no 5'-GGN(19)GG sequence, 5'-GN(20)GG or 5'-N(21)GG is also Can.

[0096] (2) The targeting site of sgRNA in HBV cccDNA is located in the ORF of S protein.

[0097] (3) Use BLAT in the UCSC database or BLAST in the NCBI database to determine whether the target sequence of the sgRNA is unique and reduce potential off-target sites.

[0098] (4) When selecting two sgRNAs to knock out genes in pairs, select paired sites separated by a certain distance (10-30 bp) on HBV cccD...

Embodiment 2

[0113] Example 2 Using CRISPR-Cas9 to specifically knock out HBV cccDNA (the sgRNA used to target HBV cccDNA is shown in the sequence table as SEQ ID NO. 30)

[0114] 1. Linearization sequence such as sequence table SEQ ID NO. 12 The indicated pGL3-U6-sgRNA plasmids.

[0115] Enzyme digestion system and conditions are as follows:

[0116] 2 μg pGL3-U6-sgRNA (400 ng / μl);

[0117] 1 μl CutSmart Buffer;

[0118] 1 μl BsaI (NEB, R0535L);

[0119] Add water to 50 μl, incubate at 37°C for 3-4 hours, shake and centrifuge at intervals to prevent droplets from evaporating onto the tube cap.

[0120] After digestion, use AxyPrep PCR Clean up Kit (AP-PCR-250) to purify and recover to 20-40 μl sterilized water.

[0121] 2. The double-stranded sgRNA oligonucleotides obtained after denaturation and annealing that can be connected to the U6 eukaryotic expression vector (the sequences of their Forward oligo and Reverse oligo are shown in the sequence table SEQ ID NO...

Embodiment 3

[0168] Example 3 Using CRISPR-Cas9 to specifically knock out HBV cccDNA (the sgRNA used to target HBV cccDNA is shown in the sequence table as SEQ ID NO. 31)

[0169] 1. Linearization sequence such as sequence table SEQ ID NO. 12 The indicated pGL3-U6-sgRNA plasmids.

[0170] Enzyme digestion system and conditions are as follows:

[0171] 2 μg pGL3-U6-sgRNA (400 ng / μl);

[0172] 1 μl CutSmart Buffer;

[0173] 1 μl BsaI (NEB, R0535L);

[0174] Add water to 50 μl, incubate at 37°C for 3-4 hours, shake and centrifuge at intervals to prevent droplets from evaporating onto the tube cap.

[0175] After digestion, use AxyPrep PCR Clean up Kit (AP-PCR-250) to purify and recover to 20-40 μl sterilized water.

[0176] 2. The double-stranded sgRNA oligonucleotides obtained after denaturation and annealing that can be connected to the U6 eukaryotic expression vector (the sequences of their Forward oligo and Reverse oligo are shown in the sequence table SEQ ID ...

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Abstract

The invention belongs to the field of genetic engineering, and particularly relates to a method for specifically knocking out hepatitis b virus cccDNA by using CRISPR-Cas9 and sgRNA for specifically targeting the hepatitis b virus cccDNA. The invention provides a method for specifically knocking out hepatitis b virus cccDNA by using CRISPR-Cas9 and sgRNA for specifically targeting the hepatitis b virus cccDNA. The sgRNA of specific targeted hepatitis b virus cccDNA prepared according to the invention can precisely target hepatitis b virus cccDNA and realize gene knockout. A preparation method is simple in steps and good in sgRNA targeting, and the knockout efficiency of a CRISPR-Cas9 system is high.

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 cccDNA by CRISPR-Cas9. Background technique [0002] Hepatitis B virus (Hepatitis B VIRUS, HBV) belongs to the hepadnaviridae family and is the pathogen that causes viral hepatitis B. About 1 / 3 of the world's people have ever been infected with HBV, and there are about 300-400 million people with chronic HBV infection, of which 25%-40% die from HBV infection-related diseases. HBV is the ninth deadliest disease in the world. my country is a high-endemic area of ​​HBV infection, and the positive rate of hepatitis B virus surface antigen (HBsAg), a marker of hepatitis B virus infection in the general population, is close to 10%. The prevention and treatment of existing viral hepatitis B patients and HBsAg carriers will still be an arduous task in the next few decades. [0003] Hepatitis B virus is also a re...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/85A61K48/00A61P31/20
Inventor 陈丽黄行许
Owner AOMIAO BIOTECH GUANGZHOU CO LTD
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