Cleavage of intracellular hepatitis B virus genomeby using of CRISPR/Cas9
10.3760/cma.j.issn.1003-9279.2016.01.014
- VernacularTitle:应用 CRISPR／Cas9系统靶向切割细胞内乙型肝炎病毒基因组的研究
- Author:
Yuqin LU
1
;
Jinchao HAN
;
Xu CONG
;
Lai WEI
;
Xiaoben PAN
Author Information
1. 100044 北京大学人民医院，北京大学肝病研究所，丙型肝炎和肝病免疫治疗北京市重点实验室
- Keywords:
Hepatitis B;
cccDNA;
CRISP/Cas9;
Genes;
Antivirus
- From:
Chinese Journal of Experimental and Clinical Virology
2016;(1):53-57
- CountryChina
- Language:Chinese
-
Abstract:
Objective To specifically cleave the intracellular HBV DNA using the clustered regularly interspaced short palindromic repeat ( CRISPR )/Cas9 genome editing technology. Methods Using of Lenti CRISPRv2 system, we designed 3 highly conserved guide RNAs ( gRNAs ) to specifically cleave HBV genome.The cleavage efficacy of CRISPR/Cas9 targeting at HBV DNA in plasmids, the episomal HBV DNA or integrated HBV DNA in chromosome was evaluated in the cell cultures of HepG2.2.15 and HepDES19, and in a model of C57BL/6 mouse infected with AAV-HBV1.3.Results Viral proteins were inhibited by 30% -50% by a single site CRISPR/Cas9 cleavagein the HepG2 cells transiently co-transfected with HBV DNA, multiple sites of cleavage significantly elevated the efficiency to 70%-90%.Compared with that of HepG2.2.15 cells, HBeAg in HepDES19 cells was inhibitedby at least three-fold higher.Conclusion Multiple sites cleavage significantly improve the efficacy of CRISPR/Cas9 systems,and this system is more efficient to cleave the extra-chromosome HBV DNA than the integrated HBV DNA in chromosomes.
