Objective:To investigate the antivirus activity of HBV targeted CRISPR-Cas9 system. Methods:Twelve sg RNAs（singleguide RNA）that target to different hepatitis B virus（HBV）genome were designed by bioinformatic screening. After transient transfection experiment in Hep G2 cell

Southern blot and quantitative real time PCR were performed to prove the anti-HBV efficiency of each psp Cas9-sg RNA plasmid. A newly published rccc DNA cell model was employed to evaluate the activity of CRISPR-Cas9 in eliminating ccc DNA. The anti-HBV effects of CRISPRCas9 in vivo were evaluated in hydrodynamic injection mice model. Results:sg RNA 5 and sg RNA 9 targeted to polymerase and core genes of HBV genome were proved to be effective by transient transfection experiment in Hep G2 cell

rc DNA were reduced significantly（P=0.000

respectively）. CRISPR-Cas9 system with sg RNA5 and sg RNA9 significantly reduced ccc DNA level in rccc DNA cell model（P=0.000

respectively）

and the short fragment deletions nearby target sites were the major mutation phenotype

50% to 60% ccc DNA possessed these deletion characteristics. Sg RNA5 and sg RNA9 containing psp Cas9 plasmid can reduce HBs Ag（P=0.000

P=0.001

respectively）

HBe Ag（P=0.000

P=0.000

respectively）and HBV DNA level（P=0.000

P=0.001

respectively）in mice serum on day 3 and day 6 after injection effectively. Immunohistochemical detection of HBc Ag in the liver tissue of mice treated with sg RNA containing CRISPR-Cas9 plasmid revealed a decrease in measurable HBc Ag in hepatocytes. Conclusion:Targeting Hepatitis B virus with CRISPR-Cas9 is an effective therapeutic approach to control the virus replication.