Dual gRNAs guided CRISPR/Cas9 system inhibits hepatitis B virus replication.
OBJECTIVE
To screen and investigate the effective gRNAs against hepatitis B virus (HBV) of genotypes A-D.


METHODS
A total of 15 gRNAs against HBV of genotypes A-D were designed. Eleven combinations of two above gRNAs (dual-gRNAs) covering the regulatory region of HBV were chosen. The efficiency of each gRNA and 11 dual-gRNAs on the suppression of HBV (genotypes A-D) replication was examined by the measurement of HBV surface antigen (HBsAg) or e antigen (HBeAg) in the culture supernatant. The destruction of HBV-expressing vector was examined in HuH7 cells co-transfected with dual-gRNAs and HBV-expressing vector using polymerase chain reaction (PCR) and sequencing method, and the destruction of cccDNA was examined in HepAD38 cells using KCl precipitation, plasmid-safe ATP-dependent DNase (PSAD) digestion, rolling circle amplification and quantitative PCR combined method. The cytotoxicity of these gRNAs was assessed by a mitochondrial tetrazolium assay.


RESULTS
All of gRNAs could significantly reduce HBsAg or HBeAg production in the culture supernatant, which was dependent on the region in which gRNA against. All of dual gRNAs could efficiently suppress HBsAg and/or HBeAg production for HBV of genotypes A-D, and the efficacy of dual gRNAs in suppressing HBsAg and/or HBeAg production was significantly increased when compared to the single gRNA used alone. Furthermore, by PCR direct sequencing we confirmed that these dual gRNAs could specifically destroy HBV expressing template by removing the fragment between the cleavage sites of the two used gRNAs. Most importantly, gRNA-5 and gRNA-12 combination not only could efficiently suppressing HBsAg and/or HBeAg production, but also destroy the cccDNA reservoirs in HepAD38 cells.


CONCLUSIONS
These results suggested that CRISPR/Cas9 system could efficiently destroy HBV expressing templates (genotypes A-D) without apparent cytotoxicity. It may be a potential approach for eradication of persistent HBV cccDNA in chronic HBV infection patients.
Jie Wang, Rui-Yang Zhang, Shan-Long Ding, Lu Long, Xiang-Mei Chen, Hui Zhuang, Feng-Min Lu, State Key Laboratory of Natural and Biomimetic Drugs, Department of Microbiology and Infectious Disease Center, School of Basic Medicine, Peking University Health Science Center, Beijing 100191, China.
Jie
J
Wang
Jie Wang, Rui-Yang Zhang, Shan-Long Ding, Lu Long, Xiang-Mei Chen, Hui Zhuang, Feng-Min Lu, State Key Laboratory of Natural and Biomimetic Drugs, Department of Microbiology and Infectious Disease Center, School of Basic Medicine, Peking University Health Science Center, Beijing 100191, China.
Zhong-Wei
ZW
Xu
Jie Wang, Rui-Yang Zhang, Shan-Long Ding, Lu Long, Xiang-Mei Chen, Hui Zhuang, Feng-Min Lu, State Key Laboratory of Natural and Biomimetic Drugs, Department of Microbiology and Infectious Disease Center, School of Basic Medicine, Peking University Health Science Center, Beijing 100191, China.
Shuang
S
Liu
Jie Wang, Rui-Yang Zhang, Shan-Long Ding, Lu Long, Xiang-Mei Chen, Hui Zhuang, Feng-Min Lu, State Key Laboratory of Natural and Biomimetic Drugs, Department of Microbiology and Infectious Disease Center, School of Basic Medicine, Peking University Health Science Center, Beijing 100191, China.
Rui-Yang
RY
Zhang
Jie Wang, Rui-Yang Zhang, Shan-Long Ding, Lu Long, Xiang-Mei Chen, Hui Zhuang, Feng-Min Lu, State Key Laboratory of Natural and Biomimetic Drugs, Department of Microbiology and Infectious Disease Center, School of Basic Medicine, Peking University Health Science Center, Beijing 100191, China.
Shan-Long
SL
Ding
Jie Wang, Rui-Yang Zhang, Shan-Long Ding, Lu Long, Xiang-Mei Chen, Hui Zhuang, Feng-Min Lu, State Key Laboratory of Natural and Biomimetic Drugs, Department of Microbiology and Infectious Disease Center, School of Basic Medicine, Peking University Health Science Center, Beijing 100191, China.
Xiao-Meng
XM
Xie
Jie Wang, Rui-Yang Zhang, Shan-Long Ding, Lu Long, Xiang-Mei Chen, Hui Zhuang, Feng-Min Lu, State Key Laboratory of Natural and Biomimetic Drugs, Department of Microbiology and Infectious Disease Center, School of Basic Medicine, Peking University Health Science Center, Beijing 100191, China.
Lu
L
Long
Jie Wang, Rui-Yang Zhang, Shan-Long Ding, Lu Long, Xiang-Mei Chen, Hui Zhuang, Feng-Min Lu, State Key Laboratory of Natural and Biomimetic Drugs, Department of Microbiology and Infectious Disease Center, School of Basic Medicine, Peking University Health Science Center, Beijing 100191, China.
Xiang-Mei
XM
Chen
Jie Wang, Rui-Yang Zhang, Shan-Long Ding, Lu Long, Xiang-Mei Chen, Hui Zhuang, Feng-Min Lu, State Key Laboratory of Natural and Biomimetic Drugs, Department of Microbiology and Infectious Disease Center, School of Basic Medicine, Peking University Health Science Center, Beijing 100191, China.
Hui
H
Zhuang
Jie Wang, Rui-Yang Zhang, Shan-Long Ding, Lu Long, Xiang-Mei Chen, Hui Zhuang, Feng-Min Lu, State Key Laboratory of Natural and Biomimetic Drugs, Department of Microbiology and Infectious Disease Center, School of Basic Medicine, Peking University Health Science Center, Beijing 100191, China.
Feng-Min
FM
Lu
D064113:CRISPR-Cas Systems; D045744:Cell Line, Tumor; D004279:DNA, Viral / Q000235:genetics* / Q000378:metabolism; D015536:Down-Regulation; D015967:Gene Expression Regulation, Viral; D005838:Genotype; D006514:Hepatitis B Surface Antigens / Q000235:genetics / Q000378:metabolism; D006513:Hepatitis B e Antigens / Q000235:genetics / Q000378:metabolism; D006515:Hepatitis B virus / Q000235:genetics* / Q000254:growth & development* / Q000378:metabolism; D006801:Humans; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics*; D014162:Transfection; D014779:Virus Replication
D004279:DNA, Viral; D006514:Hepatitis B Surface Antigens; D006513:Hepatitis B e Antigens; D000094704:RNA, Guide, CRISPR-Cas Systems
Antiviral therapy; CRISPR/Cas9; Dual gRNAs; Hepatitis B; cccDNA