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Analysis of the effect on HBV life cycle by HBV genome editing using TALEN and CRISPR/Cas9 systems
Hiromi Abe1,2, Tetsushi Sakuma3, Masataka Tsuge1,2, Nobuhiko Hiraga1,2, Michio Imamura1,2, C. Nelson Hayes1,2, Hiroshi Aikata1,2, Takashi Yamamoto3, Kazuaki Chayama1,2;
1Hiroshima University, Hiroshima, Japan; 2Liver Research Project Center, Hiroshima University, Hiroshima, Japan; 3Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Hiroshima, Japan
Background and aim: In HBV infection, interferon and other antiviral drugs can control HBV replication. However it is still difficult to eradicate HBV completely because covalently closed circular DNA (cccDNA) stably remains in the nucleus of hepato-cytes as mini-chromosomes. cccDNA works as a template for transcription for viral mRNAs after removal of nucleoside analogues and viral replication and worsening of hepatitis often occurs. Recently, new genome editing systems, TALEN and CRISPR/Cas9 systems have been developed to target specific regions of double stranded DNA sequences. The aim of this study is to investigate the efficacy of genome editing using TALEN and CRISPR/Cas9 systems to destroy HBV genomes. Method: HepG2 cells were maintained with DMEM containing 10 %FBS. Cells were seeded in 6 well-plates and co-trans-fected with 1.4xHBV genome and TALEN or CRISPR encoding plasmid in a 1:2 ratio. Three days after co-transfection, we harvested cells and culture medium to evaluate the efficacy of the genome editing by TALEN and CRISPR/Cas9 systems. The HBV DNA in culture medium was measured by qPCR. To examine viral replicative intermediates, we performed immuno-precipitation using anti-HBc antibody. After DNA purification, the core-associated HBV DNA was quantified by qPCR. TALEN plasmids were designed to target HNF4 binding sites in the core region. CRISPR plasmids were designed to target the S gene, polymerase and core region of HBV genome. Results: We designed three sets of TALEN-encoding plasmids targeting the core region and confirmed the nuclease activity by reporter-based assay. When we co-transfected 1.4xHBV genome plasmids and TALEN encoding plasmid, we did not observe any suppressive effect of TALEN. As we observed loss of protein production by TALEN expression, we thought that poor effect of TALEN was due to loss of viability in TALEN trans-fected cells. In contrast, co-transfection of plasmid of 1.4xHBV genome with CRISPR/Cas9 plasmid showed apparent reduction of HBsAg and HBeAg production compared with control plasmids. Furthermore, core associated HBV DNA declined significantly by co-transfection with CRISPR/Cas9 plasmid. Conclusion: Our results show that the CRISPR/Cas9 system is a possible candidate to target HBV DNA in infected cells. Further study is necessary to determine whether this system can reduce cccDNA in infected cells.
The following people have nothing to disclose: Hiromi Abe, Tetsushi Sakuma, Masataka Tsuge, Nobuhiko Hiraga, Michio Imamura, C. Nelson Hayes, Hiroshi Aikata, Takashi Yamamoto 作者: StephenW 时间: 2014-10-13 07:58
