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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

In vivo destruction of intrahepatic (hepatitis B virus) HBV genomes by the HBV-specific CRISPR/Cas9 system. Sera from mice receiving pAAV/HBV 1.2 together with the gRNA/Cas9 dull expression plasmid, vector (n = 4), gRNA P1 (n = 4), or gRNA XCp (n = 4), were collected after hydrodynamic injection. (a) Levels of HBsAg in sera were measured at days 2 and 7 posthydrodynamic injection as shown in the upper and bottom panel, respectively. The results were presented as individual samples with mean ± SEM. Statistical significance was calculated by the Student's t-test and indicated by asterisks (*P < 0.05, **P < 0.01). (b) Analysis of intrahepatic HBV DNA by Southern blotting. 0.1 ng of pAAV/HBV 1.2 plasmid was loaded as a positive control. Two samples from each group were analyzed in this experiment. The band intensity was determined by software ImageJ. The numbers in the bottom indicate the relative intensities of the HBV expression plasmids in each sample. (c) Results of T7E1 assay. The percentage of mismatched sequences from two of the gRNA-P1- or gRNA-XCp-treated mice (Indel %) was determined by T7E1 assay. The numbers in the bottom indicate the percentage of Indel.
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fig5: In vivo destruction of intrahepatic (hepatitis B virus) HBV genomes by the HBV-specific CRISPR/Cas9 system. Sera from mice receiving pAAV/HBV 1.2 together with the gRNA/Cas9 dull expression plasmid, vector (n = 4), gRNA P1 (n = 4), or gRNA XCp (n = 4), were collected after hydrodynamic injection. (a) Levels of HBsAg in sera were measured at days 2 and 7 posthydrodynamic injection as shown in the upper and bottom panel, respectively. The results were presented as individual samples with mean ± SEM. Statistical significance was calculated by the Student's t-test and indicated by asterisks (*P < 0.05, **P < 0.01). (b) Analysis of intrahepatic HBV DNA by Southern blotting. 0.1 ng of pAAV/HBV 1.2 plasmid was loaded as a positive control. Two samples from each group were analyzed in this experiment. The band intensity was determined by software ImageJ. The numbers in the bottom indicate the relative intensities of the HBV expression plasmids in each sample. (c) Results of T7E1 assay. The percentage of mismatched sequences from two of the gRNA-P1- or gRNA-XCp-treated mice (Indel %) was determined by T7E1 assay. The numbers in the bottom indicate the percentage of Indel.

Mentions: The HBV hydrodynamics-mouse model is a well-established model of HBV persistence.28,29 Therefore, we further examined the efficacy of the CRISPR/Cas9 system in the cleavage of HBV genome-containing plasmids in vivo by using this model. The HBV-expression vector and the CRISPR/Cas9 dual expression vectors were coinjected into the tail veins of C57BL/6 mice by hydrodynamics. We found that serum HBsAg levels were significantly lower in mice receiving HBV-specific gRNAs P1 and XCp on day 2 postinjection (Figure 5a). On day 6, serum HBsAg levels were significantly reduced only in gRNA-P1-treated mice (Figure 5a). In addition, using Southern blot analysis, we found that the levels of intrahepatic HBV-expressing vectors, an indicator for persistent HBV genomes, were significantly reduced in those mice receiving HBV-specific gRNAs P1 and XCp (Figure 5b). To confirm that this CRISPR/Cas9 system specifically cleaves the target sequences, we performed the T7E1 assay, which showed target-specific mutagenesis in mice receiving the gRNA-P1 or -XCp/Cas9 dual expression vector, but not in control mice (Figure 5c). According to the T7E1 assay, the targeted disruption occurred in 5 and 4% of intrahepatic HBV genomes in mice receiving gRNAs P1 and XCp, respectively. This result is in contrast to the efficient reduction in the levels of intrahepatic HBV-expressing vectors on Southern blot analysis. We further carried out the clonal sequencing of intrahepatic HBV DNA extracted from mice receiving gRNA P1 to demonstrate the disruption of the HBV genome by the CRISPR/Cas9 system. We analyzed 18 clones in total. Five clones (27.8%) with Indel were noted, suggesting that nonhomologous end joining (NHEJ) repair has occurred at the cleaved target sequence of gRNA P1 (Supplementary Figure S3). Taken together, our results indicated that the CRISPR/Cas9 system cleaved the HBV genome-expressing template and facilitated its clearance.


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)

In vivo destruction of intrahepatic (hepatitis B virus) HBV genomes by the HBV-specific CRISPR/Cas9 system. Sera from mice receiving pAAV/HBV 1.2 together with the gRNA/Cas9 dull expression plasmid, vector (n = 4), gRNA P1 (n = 4), or gRNA XCp (n = 4), were collected after hydrodynamic injection. (a) Levels of HBsAg in sera were measured at days 2 and 7 posthydrodynamic injection as shown in the upper and bottom panel, respectively. The results were presented as individual samples with mean ± SEM. Statistical significance was calculated by the Student's t-test and indicated by asterisks (*P < 0.05, **P < 0.01). (b) Analysis of intrahepatic HBV DNA by Southern blotting. 0.1 ng of pAAV/HBV 1.2 plasmid was loaded as a positive control. Two samples from each group were analyzed in this experiment. The band intensity was determined by software ImageJ. The numbers in the bottom indicate the relative intensities of the HBV expression plasmids in each sample. (c) Results of T7E1 assay. The percentage of mismatched sequences from two of the gRNA-P1- or gRNA-XCp-treated mice (Indel %) was determined by T7E1 assay. The numbers in the bottom indicate the percentage of Indel.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig5: In vivo destruction of intrahepatic (hepatitis B virus) HBV genomes by the HBV-specific CRISPR/Cas9 system. Sera from mice receiving pAAV/HBV 1.2 together with the gRNA/Cas9 dull expression plasmid, vector (n = 4), gRNA P1 (n = 4), or gRNA XCp (n = 4), were collected after hydrodynamic injection. (a) Levels of HBsAg in sera were measured at days 2 and 7 posthydrodynamic injection as shown in the upper and bottom panel, respectively. The results were presented as individual samples with mean ± SEM. Statistical significance was calculated by the Student's t-test and indicated by asterisks (*P < 0.05, **P < 0.01). (b) Analysis of intrahepatic HBV DNA by Southern blotting. 0.1 ng of pAAV/HBV 1.2 plasmid was loaded as a positive control. Two samples from each group were analyzed in this experiment. The band intensity was determined by software ImageJ. The numbers in the bottom indicate the relative intensities of the HBV expression plasmids in each sample. (c) Results of T7E1 assay. The percentage of mismatched sequences from two of the gRNA-P1- or gRNA-XCp-treated mice (Indel %) was determined by T7E1 assay. The numbers in the bottom indicate the percentage of Indel.
Mentions: The HBV hydrodynamics-mouse model is a well-established model of HBV persistence.28,29 Therefore, we further examined the efficacy of the CRISPR/Cas9 system in the cleavage of HBV genome-containing plasmids in vivo by using this model. The HBV-expression vector and the CRISPR/Cas9 dual expression vectors were coinjected into the tail veins of C57BL/6 mice by hydrodynamics. We found that serum HBsAg levels were significantly lower in mice receiving HBV-specific gRNAs P1 and XCp on day 2 postinjection (Figure 5a). On day 6, serum HBsAg levels were significantly reduced only in gRNA-P1-treated mice (Figure 5a). In addition, using Southern blot analysis, we found that the levels of intrahepatic HBV-expressing vectors, an indicator for persistent HBV genomes, were significantly reduced in those mice receiving HBV-specific gRNAs P1 and XCp (Figure 5b). To confirm that this CRISPR/Cas9 system specifically cleaves the target sequences, we performed the T7E1 assay, which showed target-specific mutagenesis in mice receiving the gRNA-P1 or -XCp/Cas9 dual expression vector, but not in control mice (Figure 5c). According to the T7E1 assay, the targeted disruption occurred in 5 and 4% of intrahepatic HBV genomes in mice receiving gRNAs P1 and XCp, respectively. This result is in contrast to the efficient reduction in the levels of intrahepatic HBV-expressing vectors on Southern blot analysis. We further carried out the clonal sequencing of intrahepatic HBV DNA extracted from mice receiving gRNA P1 to demonstrate the disruption of the HBV genome by the CRISPR/Cas9 system. We analyzed 18 clones in total. Five clones (27.8%) with Indel were noted, suggesting that nonhomologous end joining (NHEJ) repair has occurred at the cleaved target sequence of gRNA P1 (Supplementary Figure S3). Taken together, our results indicated that the CRISPR/Cas9 system cleaved the HBV genome-expressing template and facilitated its clearance.

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