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CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy
Published on: March 12, 2018
Removal of Integrated Hepatitis B Virus DNA Using CRISPR-Cas9
Hao Li1, Chunyu Sheng1, Shan Wang1
1Center for Infectious Disease Control, Institute of Disease Control and Prevention, Academy of Military Medical Sciences Beijing, China.
Frontiers in Cellular and Infection Microbiology
|April 7, 2017
Summary
CRISPR-Cas9 technology successfully eradicated hepatitis B virus (HBV) infection in a stable cell line by excising integrated HBV DNA and disrupting cccDNA. This breakthrough offers a potential path to a complete cure for HBV.
Area of Science:
- Molecular Biology
- Virology
- Genetics
Background:
- Hepatitis B virus (HBV) cccDNA and integrated DNA pose risks for viral reactivation and cancer.
- Current treatments like nucleoside analogs cannot eliminate these persistent HBV templates.
- CRISPR-Cas9 shows promise for disrupting HBV cccDNA, but integrated HBV DNA remains a challenge.
Purpose of the Study:
- To investigate the efficacy of CRISPR-Cas9 in completely removing integrated HBV DNA and disrupting cccDNA in a stable HBV cell line.
- To assess the long-term effects of HBV eradication on viral markers.
- To evaluate potential off-target effects of the CRISPR-Cas9 system.
Main Methods:
- Utilized a CRISPR-Cas9 system to target and excise a full-length integrated HBV DNA fragment.
- Disrupted HBV cccDNA within a stable HBV cell line.
- Monitored HBV markers (cccDNA, HBV DNA, HBsAg, HBeAg) over 10 months.
- Performed whole genome sequencing to analyze off-target mutations and confirm cell line integrity.
Main Results:
- Achieved complete excision of the integrated HBV DNA fragment and disruption of HBV cccDNA.
- Maintained undetectable levels of HBV cccDNA, HBV DNA, HBsAg, and HBeAg for over 10 months post-treatment.
- Whole genome sequencing confirmed no significant off-target effects and excluded cell contamination.
Conclusions:
- CRISPR-Cas9 system can fully eradicate HBV infection in a stable cell line by eliminating both integrated and cccDNA forms.
- This study demonstrates the potential of CRISPR-Cas9 as a powerful tool for achieving a radical or 'sterile' cure for HBV.
- Further research is warranted to translate these findings into clinical applications for HBV treatment.
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