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Specific sgRNA sequence capable of editing HBV cccDNA in targeted manner and application of specific sgRNA sequence

A sequence and targeting technology, applied in the field of gene editing, can solve the problems of stay, high specificity and low immunogenicity, and achieve the effect of improving the specificity of the C region and reducing the off-target effect.

Pending Publication Date: 2021-08-13
CHONGQING MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the lack of highly specific sgRNA sequences and low immunogenicity delivery systems, the above research often stays at the stage of cell experiments

Method used

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  • Specific sgRNA sequence capable of editing HBV cccDNA in targeted manner and application of specific sgRNA sequence
  • Specific sgRNA sequence capable of editing HBV cccDNA in targeted manner and application of specific sgRNA sequence
  • Specific sgRNA sequence capable of editing HBV cccDNA in targeted manner and application of specific sgRNA sequence

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1 Design and preparation of sgRNA targeting HBV genome

[0052] The gene sequence of the conserved region of different genotypes of HBV (HBV genome C region) was selected as the targeting sequence, and the CRISPR-sgRNA online design website (http: / / crispr.mit.edu / ) and CRISPRMultiTargeter (http: / / www.multicrispr.net / ) website for sgRNA design and screening, use NCBIBLAST to compare with the human genome (HG19), remove homologous sgRNA sequences, and improve CRISPR-Cas9 from the level of sgRNA bioinformatics design Specificity, reducing off-target effects (sgRNA sequences are synthesized by BGI).

[0053] (1) Design of sgRNAs targeting the C region of the HBV genome

[0054] Using the CRISPR-sgRNA online design website developed by Zhang Feng's laboratory ( http: / / crispr.mit .edu / , such as figure 1 shown) and CRISPR MultiTargeter (http: / / www.multicrispr.net / , such as figure 2 Shown) site design and screening of sgRNA targeting HBV genome C region (NC_00397...

Embodiment 2

[0068] Example 2 Construction of CRISPR / Cas9 system

[0069] The sgRNA and NLS-Cas9-NLS were co-incubated at room temperature for 10 min at a molar mass of 1:1 to form a complex CRISPR / Cas9.

Embodiment 3

[0070] Example 3 Research on exogenous splicing activity of CRISPR / Cas9 system

[0071] Using the HBV-CMV plasmid as a template, PCR amplified the target fragment containing the sgRNA target gene sequence, and it was verified by sequencing that the amplified fragment had no mutation; the sgRNA, Cas9 and 10× reaction buffer (200mM HEPES, 1M NaCl, 50mM MgCl2, 1mM EDTA, pH6.5) Configure the reaction system as shown in Table 3. Then 5ul of the purified PCR product was added and reacted at 37°C for 2h. Finally, the digestion efficiency was detected by 1% agarose gel electrophoresis.

[0072] Table 3: CRISPR / Cas9 System Exogenous Cutting Activity Detection Reaction System

[0073]

[0074] Then configure the reaction system with sgRNA, Cas9 and 10× reaction buffer as shown in Table 3, then add 5ul of the purified PCR product, and react at 37°C for 2h. Finally, use 1% agarose gel electrophoresis to detect the enzyme cutting efficiency (such as Figure 4 ). The in vitro activi...

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Abstract

The invention belongs to the technical field of gene editing, and particularly relates to a specific sgRNA sequence capable of editing HBV cccDNA in a targeted manner and application of the specific sgRNA sequence, wherein the sgRNA sequence is shown as Sequence NO.1 or Sequence NO.2, a designed primer pair is used for performing PCR amplification to prepare a transcription template of the sgRNA, and then, transcription is performed to obtain the sgRNA sequence. The sgRNA is used for improving the CRISPR-Cas9 system, the specificity of the CRISPR-Cas9 system of a targeted HBV genome C region is improved, and the off-target effect is reduced.

Description

technical field [0001] The invention belongs to the technical field of gene editing, and in particular relates to a specific sgRNA sequence capable of targeted editing of HBV cccDNA and an application thereof. Background technique [0002] The CRISPR / Cas system is an RNA-guided immune defense system in bacteria and archaea. The formula activates tracrRNA) and the RNA-guided Cas9 nuclease recognizes and cleaves the target site DNA. According to the type and homology of Cas protein, CRISPR system is divided into five types: I, II, III, IV and V, among which type II CRISPR / Cas9 system is the current research hotspot. In this system, Streptococcus (S.pyogenes, Sp) Cas9 is currently the most widely used gene editing tool. In 2013, Professor Zhang Feng reported for the first time in Science that CRISPR / Cas9 could work in eukaryotic cells, opening a new era of human gene editing. In 2014, Lin et al. first confirmed that CRISPR / Cas9 can target and cut the HBV genome in vitro, and...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/11C12Q1/6806C12Q1/70C12N15/63C12N9/22
CPCC12N15/1131C12N15/63C12N9/22C12Q1/6806C12Q1/701C12N2310/20
Inventor 李小松陈诗赵金秋王丹陈玲
Owner CHONGQING MEDICAL UNIVERSITY
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