iBet uBet web content aggregator. Adding the entire web to your favor.
iBet uBet web content aggregator. Adding the entire web to your favor.



Link to original content: https://pubmed.ncbi.nlm.nih.gov/28382278
Removal of Integrated Hepatitis B Virus DNA Using CRISPR-Cas9 - PubMed Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Mar 22:7:91.
doi: 10.3389/fcimb.2017.00091. eCollection 2017.

Removal of Integrated Hepatitis B Virus DNA Using CRISPR-Cas9

Hao Li  1 , Chunyu Sheng  1 , Shan Wang  1 , Lang Yang  1 , Yuan Liang  1 , Yong Huang  2 , Hongbo Liu  1 , Peng Li  1 , Chaojie Yang  1 , Xiaoxia Yang  1 , Leili Jia  1 , Jing Xie  1 , Ligui Wang  1 , Rongzhang Hao  1 , Xinying Du  1 , Dongping Xu  3 , Jianjun Zhou  4 , Mingzhen Li  5 , Yansong Sun  1 , Yigang Tong  2 , Qiao Li  6 , Shaofu Qiu  1 , Hongbin Song  1
Affiliations

Removal of Integrated Hepatitis B Virus DNA Using CRISPR-Cas9

Hao Li et al. Front Cell Infect Microbiol. .

Abstract

The presence of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) and the permanent integration of HBV DNA into the host genome confers the risk of viral reactivation and hepatocellular carcinoma. Nucleoside/nucleotide analogs alone have little or no capacity to eliminate replicative HBV templates consisting of cccDNA or integrated HBV DNA. Recently, CRISPR/Cas9 technology has been widely applied as a promising genome-editing tool, and HBV-specific CRISPR-Cas9 systems were shown to effectively mediate HBV cccDNA disruption. However, the integrated HBV DNA fragments are considered as important pro-oncogenic properties and it serves as an important template for viral replication and expression in stable HBV cell line. In this study, we completely excised a full-length 3,175-bp integrated HBV DNA fragment and disrupted HBV cccDNA in a stable HBV cell line. In HBV-excised cell line, the HBV cccDNA inside cells, supernatant HBV DNA, HBsAg, and HBeAg remained below the negative critical values for more than 10 months. Besides, by whole genome sequencing, we analyzed off-target effects and excluded cell contamination. It is the first time that the HBV infection has been fully eradicated in a stable HBV cell line. These findings demonstrate that the CRISPR-Cas9 system is a potentially powerful tool capable of promoting a radical or "sterile" HBV cure.

Keywords: CRISPR-Cas9; HBV cccDNA; hepatitis B virus; integrated HBV DNA; whole genome sequencing.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Analysis of integrated HBV DNA in the stable HBV cell line A64. (A) Integrated HBV DNA in the stable HBV A64 cell line and the gRNA target sites in the repeat region of the 1.1 HBV genome copy. (B) The 4,049-bp DNA fragment representing the 3,362-bp integrated HBV DNA (1.1 copies) plus a 687-bp pTriexHBV1.1-derived flanking sequence was efficiently amplified from cellular genomic DNA using the integrated HBV-specific primers P1 and P2. (C) PCR analysis using the A1AT and HBV S-gene primer sets conducted on total genomic DNA and circular duplex DNA to assess the effect of extraction on circular duplex DNA. (D) PCR analysis of the integrated HBV DNA or circular duplex DNA isolated from cells using the P1/P3 and S-gene primer sets. Using P1 and the HBV core region-specific primer P3, the HBV S-gene amplicons were predicted to be 542- and 572-bp, respectively. Primers P1/P3 did not amplify circular duplex DNA.
Figure 2
Figure 2
Inhibition of both HBV antigen expression and HBV replication by CRISPR-Cas9 in A64 cells after transfection. (A) DNA extracted from A64 cells transfected with gRNA-91, gRNA-69, gRNA-65, gRNA-62, and gRNA-60 was analyzed by a T7EI assay. Predicted sizes of uncut and cut bands are indicated. (B–D) Inhibition of HBsAg, HBV DNA, and HBeAg in cell culture supernatants at the indicated time points after transfection of A64 cells with gRNA-91, gRNA-69, gRNA-65, gRNA-62, and gRNA-60.
Figure 3
Figure 3
CRISPR-Cas9/gRNA-69 efficiently removed the integrated HBV genome from a stable HBV cell line. (A) Analysis of PCR amplicon lengths using a primer pair (P1 and P2) targeting the integrated HBV-flanking sequence revealed elimination of the full-length integrated HBV genome (3,173-bp), leaving one fragment (873-bp predicted segment from its flanking region). (B) Diagram showing excision of the full-length integrated HBV genome. The remaining fragment included the expected 687-bp from the integrated HBV flanking sequence and a 186-bp HBV repeat core region sequence. (C) Sanger sequencing of the remaining fragment (873-bp) showing the HBV flanking sequence (small letters, 687-bp) and the partial sequences (189 − 3 = 186-bp) of the integrated HBV repeat region B (green) and repeat region A (red) with a 3-bp deletion around the gRNA-69 targeting site (yellow-highlighted). Elimination of the full-length integrated HBV genome is indicated by a strikethrough. (D,E) The amounts of HBeAg, HBsAg and HBV DNA in cell culture supernatants and HBV cccDNA in the gRNA-empty-treated group (K-15) and gRNA-69-treated group (69-7) over 300 consecutive days. The HBsAg and HBeAg test results in the gRNA-69-treated group (69-7) were always under the negative threshold (0.05 IU/ml for HBsAg and 1 S/CO for HBeAg), and the amounts of HBV DNA and HBV cccDNA in the supernatants were always undetectable (<500 IU/ml for qPCR) in the gRNA-69-treated group (69-7). All viral markers in the gRNA-empty-treated group (K-15) remained at high levels.
Figure 4
Figure 4
Sanger sequencing of the gRNA-69/Cas9 target region both in the HBV-excised cell line 69-7 (upper half) and two ends of the full-length integrated HBV DNA in the stable HBV cell line A64 (lower half). The specific residual sequence with a three-nucleotide “GAA” deletion at the gRNA-69/Cas9 cleavage region in HBV-excised cell line 69-7 was used to distinguish it from the stable HBV cell line. The extra “GAA” deletion was marked by a red line in the stable HBV cell line A64.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Bang K. B., Kim H. J. (2014). Management of antiviral drug resistance in chronic hepatitis B. World J. Gastroenterol. 20, 11641–11649. 10.3748/wjg.v20.i33.11641 - DOI - PMC - PubMed
    1. Bloom K., Ely A., Mussolino C., Cathomen T., Arbuthnot P. (2013). Inactivation of hepatitis B virus replication in cultured cells and in vivo with engineered transcription activator-like effector nucleases. Mol. Ther. 21, 1889–1897. 10.1038/mt.2013.170 - DOI - PMC - PubMed
    1. Bonilla Guerrero R., Roberts L. R. (2005). The role of hepatitis B virus integrations in the pathogenesis of human hepatocellular carcinoma. J. Hepatol. 42, 760–777. 10.1016/j.jhep.2005.02.005 - DOI - PubMed
    1. Bréchot C. (2004). Pathogenesis of hepatitis B virus-related hepatocellular carcinoma: old and new paradigms. Gastroenterology 127(5 Suppl. 1), S56–S61. 10.1053/j.gastro.2004.09.016 - DOI - PubMed
    1. Cha C., Dematteo R. P. (2005). Molecular mechanisms in hepatocellular carcinoma development. Best Pract. Res. Clin. Gastroenterol. 19, 25–37. 10.1016/j.bpg.2004.11.005 - DOI - PubMed

Publication types