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The CRISPR/Cas9 System Facilitates Clearance of the Intrahepatic HBV Templates In Vivo.

Lin SR, Yang HC, Kuo YT, Liu CJ, Yang TY, Sung KC, Lin YY, Wang HY, Wang CC, Shen YC, Wu FY, Kao JH, Chen DS, Chen PJ - Mol Ther Nucleic Acids (2014)

Bottom Line: With the HBV-specific gRNAs, the CRISPR/Cas9 system significantly reduced the production of HBV core and surface proteins in Huh-7 cells transfected with an HBV-expression vector.Among eight screened gRNAs, two effective ones were identified.Using a hydrodynamics-HBV persistence mouse model, we further demonstrated that this system could cleave the intrahepatic HBV genome-containing plasmid and facilitate its clearance in vivo, resulting in reduction of serum surface antigen levels.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, National Taiwan University College of Medicine, Taipei, Taiwan.

ABSTRACT
Persistence of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) under current antiviral therapy is a major barrier to eradication of chronic hepatitis B (CHB). Curing CHB will require novel strategies for specific disruption of cccDNA. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system is a newly developed tool for site-specific cleavage of DNA targets directed by a synthetic guide RNA (gRNA) base-paired to the target DNA sequence. To examine whether this system can cleave HBV genomes, we designed eight gRNAs against HBV of genotype A. With the HBV-specific gRNAs, the CRISPR/Cas9 system significantly reduced the production of HBV core and surface proteins in Huh-7 cells transfected with an HBV-expression vector. Among eight screened gRNAs, two effective ones were identified. Interestingly, one gRNA targeting the conserved HBV sequence acted against different genotypes. Using a hydrodynamics-HBV persistence mouse model, we further demonstrated that this system could cleave the intrahepatic HBV genome-containing plasmid and facilitate its clearance in vivo, resulting in reduction of serum surface antigen levels. These data suggest that the CRISPR/Cas9 system could disrupt the HBV-expressing templates both in vitro and in vivo, indicating its potential in eradicating persistent HBV infection.

No MeSH data available.


Related in: MedlinePlus

Effects of the gRNAs P1 and XCp on different genotypes of (hepatitis B virus) HBV. (a) HBV-expression vector was cotransfected to Huh7 cells with either the gRNA-P1/Cas9 or gRNA-XCp/Cas9 dual expression vector alone or in combination. The lysate was collected 48 hours post-transfection. The levels of intracellular HBsAg were determined by Western blot analysis. The band intensity was measured by software ImageJ and normalized to the intensity of the mock vector (Vector) for each HBV genotype. The HBs R.I. was calculated from the results of three independent experiments and presented as mean ± SEM. Statistical significance was calculated by the Student's t-test and indicated by asterisks (*P < 0.05, **P < 0.01). N.S. means no statistical significance. Sequence alignment of gRNAs P1 (b) and XCp (c) in HBV genotypes A, B, and C. The underlined nucleotides indicated the PAM motif for the recognition of Cas9.
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fig4: Effects of the gRNAs P1 and XCp on different genotypes of (hepatitis B virus) HBV. (a) HBV-expression vector was cotransfected to Huh7 cells with either the gRNA-P1/Cas9 or gRNA-XCp/Cas9 dual expression vector alone or in combination. The lysate was collected 48 hours post-transfection. The levels of intracellular HBsAg were determined by Western blot analysis. The band intensity was measured by software ImageJ and normalized to the intensity of the mock vector (Vector) for each HBV genotype. The HBs R.I. was calculated from the results of three independent experiments and presented as mean ± SEM. Statistical significance was calculated by the Student's t-test and indicated by asterisks (*P < 0.05, **P < 0.01). N.S. means no statistical significance. Sequence alignment of gRNAs P1 (b) and XCp (c) in HBV genotypes A, B, and C. The underlined nucleotides indicated the PAM motif for the recognition of Cas9.

Mentions: We also examined the effects of the conserved gRNA XCp on the HBV genomes of genotypes B and C. Our data demonstrated that gRNA-XCp/Cas9 was equally effective in suppressing the viral protein expression of all three genotypes of HBV. In contrast, the most effective gRNA P1 against genotype A of HBV did not significantly suppress the viral protein expression of genotype B, although it significantly suppressed that of genotype C (Figure 4a). Nevertheless, the suppressive effect of gRNA P1 on viral protein expression of genotype C is much less than that of gRNA XCp (P < 0.05), whereas both gRNAs exhibited a similar inhibitory effect on genotype A. Analysis of the target sequences of gRNA P1 in the HBV genomes of these three genotypes revealed that the HBV genomes of genotypes B and C had a single-nucleotide polymorphism in the PAM, although they had the perfectly matched 20-bp target sequence (Figure 4b). In contrast, the target sequence and PAM of gRNA XCp are exactly the same in all three genotypes (Figure 4c). This result indicates the importance of PAM for genome cleavage by the CRISPR/Cas9 system, and also provides further evidence that the effects of gRNAs P1 and XCp are mediated by the CRISPR/Cas9 system.


The CRISPR/Cas9 System Facilitates Clearance of the Intrahepatic HBV Templates In Vivo.

Lin SR, Yang HC, Kuo YT, Liu CJ, Yang TY, Sung KC, Lin YY, Wang HY, Wang CC, Shen YC, Wu FY, Kao JH, Chen DS, Chen PJ - Mol Ther Nucleic Acids (2014)

Effects of the gRNAs P1 and XCp on different genotypes of (hepatitis B virus) HBV. (a) HBV-expression vector was cotransfected to Huh7 cells with either the gRNA-P1/Cas9 or gRNA-XCp/Cas9 dual expression vector alone or in combination. The lysate was collected 48 hours post-transfection. The levels of intracellular HBsAg were determined by Western blot analysis. The band intensity was measured by software ImageJ and normalized to the intensity of the mock vector (Vector) for each HBV genotype. The HBs R.I. was calculated from the results of three independent experiments and presented as mean ± SEM. Statistical significance was calculated by the Student's t-test and indicated by asterisks (*P < 0.05, **P < 0.01). N.S. means no statistical significance. Sequence alignment of gRNAs P1 (b) and XCp (c) in HBV genotypes A, B, and C. The underlined nucleotides indicated the PAM motif for the recognition of Cas9.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4221598&req=5

fig4: Effects of the gRNAs P1 and XCp on different genotypes of (hepatitis B virus) HBV. (a) HBV-expression vector was cotransfected to Huh7 cells with either the gRNA-P1/Cas9 or gRNA-XCp/Cas9 dual expression vector alone or in combination. The lysate was collected 48 hours post-transfection. The levels of intracellular HBsAg were determined by Western blot analysis. The band intensity was measured by software ImageJ and normalized to the intensity of the mock vector (Vector) for each HBV genotype. The HBs R.I. was calculated from the results of three independent experiments and presented as mean ± SEM. Statistical significance was calculated by the Student's t-test and indicated by asterisks (*P < 0.05, **P < 0.01). N.S. means no statistical significance. Sequence alignment of gRNAs P1 (b) and XCp (c) in HBV genotypes A, B, and C. The underlined nucleotides indicated the PAM motif for the recognition of Cas9.
Mentions: We also examined the effects of the conserved gRNA XCp on the HBV genomes of genotypes B and C. Our data demonstrated that gRNA-XCp/Cas9 was equally effective in suppressing the viral protein expression of all three genotypes of HBV. In contrast, the most effective gRNA P1 against genotype A of HBV did not significantly suppress the viral protein expression of genotype B, although it significantly suppressed that of genotype C (Figure 4a). Nevertheless, the suppressive effect of gRNA P1 on viral protein expression of genotype C is much less than that of gRNA XCp (P < 0.05), whereas both gRNAs exhibited a similar inhibitory effect on genotype A. Analysis of the target sequences of gRNA P1 in the HBV genomes of these three genotypes revealed that the HBV genomes of genotypes B and C had a single-nucleotide polymorphism in the PAM, although they had the perfectly matched 20-bp target sequence (Figure 4b). In contrast, the target sequence and PAM of gRNA XCp are exactly the same in all three genotypes (Figure 4c). This result indicates the importance of PAM for genome cleavage by the CRISPR/Cas9 system, and also provides further evidence that the effects of gRNAs P1 and XCp are mediated by the CRISPR/Cas9 system.

Bottom Line: With the HBV-specific gRNAs, the CRISPR/Cas9 system significantly reduced the production of HBV core and surface proteins in Huh-7 cells transfected with an HBV-expression vector.Among eight screened gRNAs, two effective ones were identified.Using a hydrodynamics-HBV persistence mouse model, we further demonstrated that this system could cleave the intrahepatic HBV genome-containing plasmid and facilitate its clearance in vivo, resulting in reduction of serum surface antigen levels.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, National Taiwan University College of Medicine, Taipei, Taiwan.

ABSTRACT
Persistence of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) under current antiviral therapy is a major barrier to eradication of chronic hepatitis B (CHB). Curing CHB will require novel strategies for specific disruption of cccDNA. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system is a newly developed tool for site-specific cleavage of DNA targets directed by a synthetic guide RNA (gRNA) base-paired to the target DNA sequence. To examine whether this system can cleave HBV genomes, we designed eight gRNAs against HBV of genotype A. With the HBV-specific gRNAs, the CRISPR/Cas9 system significantly reduced the production of HBV core and surface proteins in Huh-7 cells transfected with an HBV-expression vector. Among eight screened gRNAs, two effective ones were identified. Interestingly, one gRNA targeting the conserved HBV sequence acted against different genotypes. Using a hydrodynamics-HBV persistence mouse model, we further demonstrated that this system could cleave the intrahepatic HBV genome-containing plasmid and facilitate its clearance in vivo, resulting in reduction of serum surface antigen levels. These data suggest that the CRISPR/Cas9 system could disrupt the HBV-expressing templates both in vitro and in vivo, indicating its potential in eradicating persistent HBV infection.

No MeSH data available.


Related in: MedlinePlus