RU2652899C1 - Rna-conductors to suppress the replication of hepatitis b virus and for the elimination of hepatitis b virus from host cell - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, namely, to genetic engineering. RNA-conductor (RNA guide) has been described for suppression the expression of the hepatitis B virus in a host cell and for eliminating HBV DNA from a host cell, wherein said RNA-conductor comprises a first nucleotide sequence selected from SEQ ID NO: 1 and SEQ ID NO: 2 and the second nucleotide sequence of SEQ ID NO: 5, flanking the first sequence from the 3 'end and representing an RNA-pin, and said first sequence is capable of binding to a target highly conserved region of the hepatitis B virus genome, and said second sequence contains a RAM-motive capable of being recognized by the RNA-directed Cas9 Streptococcus pyogenes DNA endonuclease, with the assurance of eliminating the hepatitis B virus. Also, an RNA transducer for suppressing the expression of the hepatitis B virus in the host cell and for eliminating HBV DNA from host cell is described, wherein said RNA-conductor comprises a first nucleotide sequence selected from SEQ ID NO: 3 and SEQ ID NO: 4, and the second nucleotide sequence of SEQ ID NO: 6, flanking the first sequence from the 3'-end and representing an RNA-hairpin, said first sequence being capable of binding to the target highly conserved region of the hepatitis B virus genome, and said second sequence is capable of being recognized by the RNA-directed Cas9 Streptococcus thermophilus DNA assay to ensure the elimination of the hepatitis B virus. Inventions can be used in CRISPR-Cas9 systems to affect highly conserved regions of the hepatitis B virus (HBV) genome, followed by elimination of HBV virus DNA, including annular covalently closed DNA (accDNA) of HBV, from a host cell, such as a mammalian cell (eg, human), in particular from human hepatocyte, as well as to suppress replication of the hepatitis B virus.

EFFECT: in comparison with prior art RNA conductors used in CRISPR-Cas9 systems to effect HBV DNA, the RNA conductors of the invention provide enhanced effect of such systems on highly conserved regions of the hepatitis B virus genome and shorten the timing of elimination of viral DNA from the host cell.

10 cl, 8 dwg, 2 tbl, 3 ex

Description

The invention relates to the field of medicine and genetic engineering, and in particular to a number of RNA-conductors (RNA directing) agents that can be used in CRISPR-Cas9 systems to influence highly conserved regions of the hepatitis B virus genome (HBV) with subsequent elimination of HBV DNA , including circular covalently closed DNA (cccDNA, cccDNA) of HBV, from a host cell, such as a mammalian (e.g. human) cell, in particular from human hepatocyte, as well as to suppress hepatitis B virus replication. By using the RNA conductors used in CRISPR-Cas9 systems for influencing HBV DNA, the RNA conductors of the invention provide an enhanced effect of such systems on highly conserved regions of the hepatitis B virus genome and a reduction in the elimination of viral DNA from the host cell.

Hepatitis B virus (HBV) is a highly contagious virus that can infect humans and cause acute or chronic hepatitis B. Cases of viral hepatitis B infection are reported worldwide, with the highest prevalence in the countries of the Middle East, the Middle East, Asia, the Caribbean, Africa and South America. According to the WHO, 350 million new cases of viral hepatitis B infection are reported annually, of which 1 million die every year from its effects, cirrhosis of the liver or hepatocellular carcinoma (HCC) (Custer B et al., 2004; Wright TL, 2006). In the Russian Federation, as in Western Europe, the USA, Canada, Australia, HBV vaccination measures have significantly reduced the incidence of acute hepatitis B, however, more than forty thousand new cases of chronic HBV infection, the main cause of cirrhosis and liver cancer, are recorded in our country every year (hepatocellular carcinoma), and an absolutely large proportion of hepatitis B cases occur in the young able-bodied population (90%). The prevalence of viral hepatitis B in the Russian Federation is 2% of the total population of the country, that is, about 3 million people. The incidence of hepatitis B virus is one of the most serious problems of Russian health care. (Federal Service for Supervision of Consumer Rights Protection and Human Well-Being, State Report “On the State of Sanitary and Epidemiological Well-Being of the Population in the Russian Federation in 2013”, 06/24/2014, http://rospotrebnadzor.ru/documents/details.php? ELEMENT_ID = 1984).

The acute form of HBV infection is most often asymptomatic and is characterized by spontaneous elimination of the virus. However, in some cases, an acute form of infection can flow into a chronic one. As shown in the literature, a chronic form of infection (CHB) develops in 95% of newborns, in 20-30% of children aged 1 to 5 years, and in less than 5% of adults (Trépo, et al. Lancet, 2014, 384 (9959 ): 2053-63). The literature has shown that 1-2% of adults with chronic hepatitis B develop liver cirrhosis, 2-5% develop hepatocellular carcinoma (Liaw et al, Hepatology, 1988, 8: 493-496; Fattovich et al, Gastroenterology, 2004; 127: S35-S50). In children suffering from HBV, the risk of developing hepatocellular carcinoma is significantly increased: it is shown that it develops in 25-50% of children infected with HBV. In general, it has been shown that approximately 40% of people with chronic hepatitis B develops cirrhosis, hepatocellular carcinoma, or liver failure sooner or later (Perz et al, Journal of Hepatology, 2006; 45: 529-538).

As the closest analogue of the present invention, the work of Kennedy E.M., et al. (“Suppression of hepatitis B virus DNA accumulation in chronically infected cells using a bacterial CRISPR / Cas RNA-guided DNA endonuclease” // Virology, 2015, 476: 196-205, URL: https: //www.ncbi.nlm.nih .gov / pmc / articles / PMC4323668), in which the use of RNA conductors together with Cas9 Streptococcus pyogenes proteins can affect various stages of the hepatitis B virus life cycle, which allows not only to reduce the total level of HBV viral DNA by about 1000 times, but also reduce the level of HBV ccDNA by about 10 times; the use of RNA conductors in conjunction with Cas9 proteins also allows inactivation of residual HBV csDNA.

The objective of the invention is the development of new tools - RNA conductors (guide RNA), which can be used in CRISPR-Cas9 systems with Cas9 proteins, such as Cas9 proteins Streptococcus pyogenes or Streptococcus thermophilus, to eliminate DNA and pregenomic RNA of hepatitis B virus from the cell the host (for example, from a eukaryotic cell, such as a mammalian hepatocyte cell or, more specifically, a human hepatocyte cell), and exposure to various stages of the hepatitis B virus life cycle (including those capable of chronic persistence in rganizme host and lead to development of an infected person cirrhosis and / or hepatocellular carcinoma). The invention provides an increase in the effectiveness of treatment of acute and chronic viral hepatitis B, as well as an increase in the effectiveness of the prevention of cirrhosis of the liver and hepatocellular carcinoma caused by chronic persistence of the ring covalently closed DNA of hepatitis B virus in the host body.

Achievable technical result is comprehensive and includes:

- increase the degree of elimination of pregenomic RNA (pgRNA, pgRNA) of the HBV virus - a correlate of active transcription in HBV infection;

- reducing the duration of the course of therapy required to eliminate HBV ccDNA;

- an increase in the elimination of HBV ccDNA, a correlate of chronic HBV persistence in the host, with an outcome in hepatocellular carcinoma and / or cirrhosis;

- reduction of elimination of HBV ccDNA from hepatocytes;

- reduction of secreted HBV DNA;

- reduction of surface antigen HBV (HBsAg);

- a decrease in core protein, a component of virions and a transcriptional regulator of cccDNA;

- reduction of X protein of HBV, the main pro-oncogenic factor, one of the key components of the development of fibrosis and HCC;

- expanding the arsenal of tools that can be used in the CRISPR-Cas9 system to affect highly conserved regions of the hepatitis B virus genome to eliminate various stages of the hepatitis B virus life cycle, including HBV pgRNA and HBV ccDNA.

The problem is solved, and the technical result is achieved by creating an RNA conductor molecule to suppress the expression of hepatitis B virus in the host cell and to eliminate hepatitis B virus DNA from the host cell, where the specified RNA conductor contains the first nucleotide sequence selected from SEQ ID NO: 1 (TCCGCAGTATGGATCGGCAG) and SEQ ID NO: 2 (GCAGATGAGAAGGCACAGAC), and the second nucleotide sequence of SEQ ID NO: 5, flanking the first sequence from the 3'-end and representing an RNA hairpin, and this first sequence is able to bind target highly conserved region of the hepatitis B virus genome, and said second sequence contains a PAM motif that can be recognized by Cas9 Streptococcus pyogenes RNA-guided DNA endonuclease to ensure hepatitis B virus elimination.

According to preferred embodiments, the specified technical result is also achieved by the fact that:

- RNA-guided DNA endonuclease Cas9 Streptococcus pyogenes recognizes a DNA sequence containing the NGM PAM motif;

- the host cell is a mammalian cell infected with hepatitis B virus, or a mammalian cell in which hepatitis B virus persists;

- Hepatitis B virus DNA is circular covalently closed DNA (ccDNA).

The RNA conductor molecule according to claim 1, characterized in that the hepatitis B virus has a genotype that is selected from the group comprising genotypes A, B, C, D, E, F, G and H.

The problem is also solved, and the technical result is achieved by creating an RNA conductor molecule to suppress the expression of hepatitis B virus in the host cell and to eliminate the hepatitis B virus DNA from the host cell, where the specified RNA conductor contains the first nucleotide sequence selected from SEQ ID NO : 3 (GGCGGGGTTTTTCTTGTTGA) and SEQ ID NO: 4 (GGCACTAGTAAACTGAGCCA), and the second nucleotide sequence of SEQ ID NO: 6, flanking the first sequence from the 3'-end and representing an RNA hairpin, and the indicated first sequence is capable of binding sya the target highly conserved portion of the genome of the virus of hepatitis B, and said second sequence is capable recognized by an RNA directed DNA endonuclease Cas9 Streptococcus thermophilus with elimination of hepatitis C virus software B.

According to preferred embodiments, the specified technical result is also achieved by the fact that:

- RNA-driven DNA endonuclease Cas9 Streptococcus thermophilus recognizes a DNA sequence containing the PAM motif NNAGAAW;

- the host cell is a mammalian cell infected with hepatitis B virus, or a mammalian cell in which hepatitis B virus persists;

- DNA of hepatitis B virus is a circular covalently closed DNA (kkzDNA);

- the hepatitis B virus has a genotype that is selected from the group comprising genotypes A, B, C, D, E, F, G and H.

The invention is also illustrated by the drawings.

In FIG. Figure 1 shows a decrease in HBV transcription during co-transfection of S. thermophiles CRISPR / Cas9 systems and plasmid 1.1merHBV in HepG2 cells already 3 days after transfection. RNA conductor SEQ ID NO: 4 (St4) reduces pgRNA and S-RNA (S-RNA of HBV) by 87% and 62%, respectively, while SEQ ID NO: 3 (St3) reduces HBV transcription only by pgRNA by 69%.

In FIG. Figure 2 shows the antiviral effect of S. thermophiles CRISPR / Cas9 systems with RNA conductors SEQ ID NO: 3 (St3) and SEQ ID NO: 4 (St4). Highly efficient nucleofection of a model cell of chronic HBV infection HepG2-1.1merHBV, plasmids encoding StCas9-T2A-Blasticidin and PCR products with U6 promoter and St3 and St4 RNA conductors, followed by negative selection of untransfected cells. St3 and St4 lead to a significant decrease in all indicators of the viral cycle (pgRNA by 56% and 61%, S-RNA by 63% and 64%, secreted by HBsAg at 0.45 IU / ml and 1.65 IU / ml, secreted by HBV DNA by 59% and 53%, cccDNA by 60% and 74% for St3 and St4, respectively).

In FIG. 3 shows the nucleofection of HepG2-1.1merHBV cells. In FIG. 3A shows the results of FACS analysis of nucleated cells via the green FITC-A channel; in FIG. 3B shows immunofluorescence photographs of nucleophased cells in a bright field (left) and on the FITC-A channel (right). The nucleofection efficiency reproducibly is 85 ± 2%.

In FIG. Figure 4 shows a comparison of the antiviral efficacy of fifty CRISPR / Cas9 RNA conductors. On the model of co-transfection of HepG2 cells with CRISPR / Cas9 systems and an HBV-cycle plasmid (1.1merHBV), the antiviral activity of fifty individual RNA conductors was evaluated, including Sp37 (SEQ ID NO: 2), Sp31, Sp20 (SEQ ID NO : 1), Sp15, Sp12, Sp11, Sp9 and Sp8 showed high efficiency in reducing HBV transcription.

In FIG. 5 shows the antiviral effect of S.pyogenes CRISPR / Cas9. On the 7th day after nucleofection of HepG2-1.1merHBV cells with a plasmid encoding Cas9-T2A-Blasticidin and PCR products with a U6 promoter and Sp20 or Sp37 RNA conductor, the HBV pgRNA transcription is reduced by 85% and 86%, S -RNA by 70% and 43% and ccDNA by 79% and 82% for Sp20 (SEQ ID NO: 1) and Sp37 (SEQ ID NO: 2), respectively.

In FIG. Figure 6 shows CRISPR / Cas9 transfection with efficient RNA conductors reduces HBcAg production of HBV. During co-transfection of HepG2 cells with CRISPR / Cas9 systems with a plasmid that starts the HBV cycle, the production of HBV core antigen, a protein involved in the assembly of virions and transcription of cccDNA, is reduced (image shows the action of Sp20). Immunocytochemical staining of HepG2 cells, red channel (594 nm) HBcAg; blue channel (365 nm) Hoechst33342, cell nuclei, Merged - image overlay.

In FIG. Figure 7 shows CRISPR / Cas9 transfection with efficient RNA conductors reduces HBxAg HBV production. Co-transfection of HepG2 cells reduces the production of HBx HBV protein, the main HBV pro-oncogenic protein, which plays a key role in the malignant transformation of hepatocytes and the development of hepatocellular carcinoma. Immunocytochemical staining of HepG2 cells, red channel (594 nm) HBxAg; blue channel (365 nm) Hoechst33342, cell nuclei, Merged - image overlay.

In FIG. Figure 8 shows the insertion of breaks into HBV ccDNA. HBV ccDNA was isolated on the 7th day after nucleofection using Hirt extraction and treatment of isolates with the enzyme Plasmid-safe ATP-dependent DNase, which destroys all types of DNA except for ring forms. The new generation sequencing using MiSeq was performed on amplicons obtained using specific primers from the site encompassing the nucleolytic rupture site. As can be seen from the drawing, the Sp20 RNA conductor with the plasmid encoding S.pyogenes Cas9 introduces numerous mutations (deletions are shown in the drawing), including deletions, insertions, inversions, recombinations, and SNPs in the gap zone.

The following are examples of the invention, which illustrate, but do not cover all possible embodiments of the invention and do not limit the invention.

Examples of carrying out the invention

Example 1

Four types of CRISPR / Cas9 systems obtained from 4 types of bacterial organisms were used in the experiments: Cas9 Streptococcus pyogenes (SpCas9), Cas9 Streptococcus thermophiles (StCas9), Cas9 Neisseria meningitides (NmCas9) and Cas9 Francicella novicida (FnCas9). The difference between these systems lies in the different consensus of the PAM region: NGG for SpCas9 and FnCas9 (while SpCas9 can only cleave DNA, while Rn activity was also shown for FnCas9), NmCas9 with PAM NNNNGATT and NNAGAAW for StCas9.

The first stage of this work was the selection of target sequences in ccDNA to create RNA conductors, for this purpose we used modern in silico algorithms and publicly available programs, including BroadInstitute (https://portals.broadinstitute.org/gpp/public / analysis-tools / sgrna-design) and Center for Organismal Studies, Heidelberg (http://crispr.cos.uni-heidelberg.de/). A list of sites on the HBV genome with a theoretically calculated probability of their cleavage in conservative and relatively conservative parts of the genome was compiled.

The second stage is the synthesis of PCR products containing the U6 promoter and the region corresponding to the RNA conductor using two-stage mutagenic PCR (Cong L et al., 2013). Intermediates and PCR products were purified on Qiagen columns (QIAquick gel extraction kit) after excision from the gel. The purity and concentration of PCR products was evaluated by optical light absorption on a Nanodrop 2000 spectrophotometer. To verify the exact match of the obtained nucleotide sequences with the declared sequences of PCR products, clonal sequencing was performed. PCR products were obtained in reactions with high precision Q5 polymerase (New England Biolabs).

The third stage is to check the efficiency of cutting selected sites in cell cultures of in vitro models of chronic HBV infection. At the heart of this phase are two approaches.

The first step is co-transfection of the HepG2 human hepatoma cell line with vectors encoding the HBV gene under the inducible tet-on promoter (HBV genotype D, 1.1mer gene, provided by Dieter Glebe, Giessen University), plasmids encoding the Cas9 proteins (Addgene # 63592 - SpCas9; Addgene # 48669 - StCas9; Addgene # 48670 - NMCas9; Addgene # 68705 - FnCas9) and synthesized PCR products encoding an RNA conductor. Cells were co-transfected with polyethyleneimine reagent or Lipofectamine 2000 reagent, HBV transcription was activated for 24 hours by treatment with doxycycline (100 ng / ml). Evaluation of the antiviral effect of CRISPR / Cas9 systems was performed after 3 days according to the main transcripts of HBV ccDNA - pregenomic HBV RNA and HBV S-RNA by real-time PCR using a pair of specific primers and probes after isolation of nucleic acids, treatment with type I DNase without RNase activity and reverse transcription.

The second step is nucleofection of a model of chronic HBV infection, a transgenic human cell line with an insert 1.1mer of the HBV genome under an inducible tet-on promoter. HepG2-1.1merHBV cells reproduce a complete HBV replication cycle, including the formation of a full-fccDNA, HBsAg secretion, etc. Nucleofection was performed on a Lonza instrument using Amaxa 4D Nucleofector kits (V4XC-2024). The nucleofection mixture included: (1) a vector encoding a Cas9 protein with an antibiotic resistance protein to blastidin and (2) a PCR product encoding an RNA conductor. Cells were nucleoficated, activated immediately after nucleofection for 24 hours in doxycycline medium (100 ng / ml). The efficiency of nucleofection according to flow cytofluorimetry was 80-85%. To remove non-transfected cells, negative selection was performed by incubating the cells in complete medium with a selected concentration of blasticidin. Cell removal and isolation of nucleic acids was performed on the 5th day after nucleofection. The antiviral effect of CRISPR / Cas9 systems was evaluated by expression of pregenomic HBV RNA, HBV S-RNA, amounts of secreted HBsAg, secreted HBV DNA, and relative amount of cccDNA. Residual ccDNA matrices in experimental samples with CRISPR / Cas9 systems were used to amplify the nucleolytic cleavage site and sequencing of PCR products by NGS (MiSeq). Mutation frequency was calculated in the program Geneious 6.1.8.

Example 2

Both types of systems (S.thermophiles and S.pyogenes) require an RNA conductor consisting of a target region adjacent to the PAM sequence (NGG and NNAGAAW, respectively) and a hairpin recognized by Cas9 protein.

In the screening model — co-transfection of HepG2 cells with a plasmid encoding a Cas9 protein, a plasmid 1.1merHBV, which starts the virus cycle, and PCR products encoding the corresponding RNA conductors, RNA conductors with the highest antiviral values were selected for the effect on the reduction of HBV transcription. (see Figs. 1, 3). Further analysis revealed RNA conductors in highly conserved regions of the HBV genome (see Table 1), which allows the use of these RNA conductors for the treatment of most patients with chronic hepatitis B, regardless of genotype or suitable for most genotypes.

Table 1. The percentage of absolute (exact) matches of RNA conductor sequences, taking into account the PAM recognition sequences for S.pyogenes CRISPR / Cas9 (NGG) and S.thermophiles CRISPR / Cas9 (NNAGAAW) among all HBV genotypes (A-H). The percentage was calculated in the Geneious 6.1.8 program with genome-wide HBV sequences taken from the GenBank database. All selected RNA conductors, taking into account PAM-recognition regions, are located in highly conserved regions that are identical for most genotypes and the absolute majority of subtypes within each genotype.

RNA conductor A (794),% B (1450),% C (1830),% D (882),% E (250),% F (227),% G (36),% H (26),% Antiviral activity (pgRNA,%) Sp20 98.7 83.7 90.3 91.6 86.4 96.5 one hundred 88.5 90.7 Sp37 97.1 81.1 91.5 97.8 75.6 1.8 11.1 0 87.4 St3 95.6 3.0 88.7 93.5 0 0.9 97.2 0 69.8 St4 98.6 97.2 92.7 97.3 98 0.9 one hundred 3.8 86.6

Nucleofection experiments on a model of chronic HepG2-1.1merHBV infection (see Figs. 2, 3, 5) revealed that S.thermophiles and S.pyogenes CRISPR / Cas9 reduce all HBV cycle parameters: transcription (pgRNA, S-RNA), and most importantly, the key component of the viral cycle responsible for the chronization of infection, circular covalently closed DNA (reduction of ccDNA by 60% -74% for S.thermophiles and 79% -82% for S.pyogenes CRISPR / Cas9). It is worth noting that in the prototype and other objects of the prior art, the reduction of ccDNA occurs by 67% (Kennedy EM, et al. “Suppression of hepatitis B virus DNA accumulation in chronically infected cells using a bacterial CRISPR / Cas RNA-guided DNA endonuclease” / / Virology. 2015, 476: 196-205) and 71% (Ramanan V., et al. “CRISPR / Cas9 cleavage of viral DNA efficiently suppresses hepatitis B virus” // Scientific Reports. 2015; 5: 10833), which is worse indicators to reduce kczDNA, which was achieved by the authors of the present invention - 79-82%.

In addition, in both of the aforementioned works, the results were obtained by the 13th day or by the 21st day after the introduction of CRISPR / Cas9 systems, while under the conditions of the present invention all the results were obtained on the 7th day, and on one of the best models of chronic hepatitis HepG2-1.1merHBV cells that reproduce a full replication cycle and are closest in characteristics to the natural HBV cycle. In general, this suggests a significantly more pronounced antiviral effect of the selected RNA conductors. Moreover, unlike all previously published data in the prior art, the S.thermophiles CRISPR / Cas9 systems for the chronic hepatitis model were first used in the present invention. It is known that S.thermophiles CRISPR / Cas9 have a smaller size, greater specificity of action and less likelihood of introducing non-targeted breaks in comparison with the classic S.pyogenes Cas9 protein, which is associated with the structural features of the S.thermophiles protein and a longer RAM (NNAGAAW - seven letters against NGG - three letters in S.pyogenes Cas9) (Müller M., et al. “Streptococcus thermophilus CRISPR-Cas9 Systems Enable Specific Editing of the Human Genome” // Molecular Therapy. 2016, 24 (3): 636-644) .

Example 3

Data were also obtained on a decrease in the secretion of HBsAg, HBV DNA and a decrease in the intensity of cell staining for HBcAg HBV and HBxAg (Fig. 2, 5, 7). Using the new generation sequencing method, it was demonstrated that the remaining ccDNA matrices contain inde mutations and other polymorphisms at the sites of RNA conductors (Fig. 6, Table 2), which indicates the direct nucleolytic effect of CRISPR / Cas9 systems on ccdDNA.

Table 2. A summary table on the introduction of mutations in ccDNA under the influence of CRISPR / Cas9 systems with RNA conductor Sp20. Results were obtained on cccDNA on day 7 after nucleofection of the CRISPR / Cas9 system. Amplicons covering the nucleolytic cleavage site were obtained with cccDNA and sequenced by the NGS method on a MiSeq sequencer (97.079 reads). The results were processed in the program Geneious 6.1.8.

Deletions (1-4 nts),
% Deletions (5-10 nts),
% Deletions
(13 nts)
% General deletions
% Insertions
(1-4 nts)
% General insertions
% SNP
% Option Frequency 17.40 1.12 0.50 19.10 0.35 35.10 0.49 Reading Frame Mutation Frequency 10,20 1.10 0.50 11.80 0.21 21.10 0.49 The proportion of mutations with shift reading frames 61.80 60.10 one hundred

Thus, inhibition of the viral cycle and insertion of mutations into ccDNA matrices that have not undergone nucleolytic degradation are associated with the action of the selected CRISPR / Cas9 systems with the indicated RNA conductors. Unlike siRNA and miRNA technologies, which use short RNAs to interfere with HBV transcripts and do not directly affect cccDNA, the effect of CRISPR / Cas9 systems is associated with the direct nucleolytic effect of the Cas9 protein on the target (ccDNA). HBV is a genetically heterogeneous virus, therefore, the production of highly efficient RNA conductors directing Cas9 protein to conserved HBV sites is one of the key steps in creating a promising antiviral agent. The targets of the obtained RNA conductors are located in highly conserved regions (the absolute coincidence rate, taking into account adjacent PAM sequences, is higher than 90% for most HBV genotypes), cause cleavage of the target target (cccDNA), or introduce numerous mutations and have a powerful antiviral effect on all the models used.

Claims (10)

1. An RNA conductor molecule for suppressing the expression of hepatitis B virus in a host cell and for eliminating hepatitis B virus DNA from a host cell, wherein said RNA conductor contains a first nucleotide sequence selected from SEQ ID NO: 1 and SEQ ID NO: 2, and the second nucleotide sequence of SEQ ID NO: 5, flanking the first sequence from the 3´ end and representing an RNA hairpin, the first sequence being able to bind to the target highly conserved region of the hepatitis B virus genome, and the second PAM contains been consistent motif ability to recognize an RNA directed DNA endonuclease Cas9 Streptococcus pyogenes with elimination of hepatitis C virus software B. 2. The RNA conductor molecule according to claim 1, characterized in that the RNA-guided DNA endonuclease Cas9 Streptococcus pyogenes recognizes a DNA sequence containing the NGM PAM motif. 3. The RNA conductor molecule according to claim 1, characterized in that the host cell is a mammalian cell infected with hepatitis B virus or a mammalian cell in which hepatitis B virus persists. 4. The RNA conductor molecule according to claim 1, characterized in that the hepatitis B virus DNA is a circular covalently closed DNA (ccDNA) DNA. 5. The RNA conductor molecule according to claim 1, characterized in that the hepatitis B virus has a genotype that is selected from the group comprising genotypes A, B, C, D, E, F, G and H. 6. An RNA conductor molecule for suppressing the expression of hepatitis B virus in a host cell and for eliminating hepatitis B virus DNA from the host cell, wherein said RNA conductor contains the first nucleotide sequence selected from SEQ ID NO: 3 and SEQ ID NO: 4, and the second nucleotide sequence of SEQ ID NO: 6, flanking the first sequence from the 3´-end and representing an RNA hairpin, the indicated first sequence being able to bind to the target highly conserved region of the hepatitis B virus genome, and the second research can be recognized by Cas9 Streptococcus thermophilus RNA-guided DNA endonuclease to eliminate hepatitis B virus. 7. The RNA conductor molecule of claim 6, wherein the RNA-guided Cas9 Streptococcus thermophilus DNA endonuclease recognizes a DNA sequence containing the NNAGAAW PAM motif. 8. The RNA conductor molecule according to claim 6, wherein the host cell is a mammalian cell infected with hepatitis B virus or a mammalian cell in which hepatitis B virus persists. 9. The RNA conductor molecule according to claim 6, characterized in that the hepatitis B virus DNA is a circular covalently closed DNA (ccDNA) DNA. 10. The RNA conductor molecule according to claim 6, characterized in that the hepatitis B virus has a genotype that is selected from the group comprising genotypes A, B, C, D, E, F, G and H.

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