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Updated: Mar 20, 2026
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Using Next Generation Sequencing to Identify Mutations Associated with Repair of a CAS9-induced Double Strand Break Near the CD4 Promoter
Published on: March 31, 2022
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Complete Spectrum of CRISPR/Cas9-induced Mutations on HBV cccDNA
1Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania, USA.
Summary
The CRISPR/Cas9 system effectively inactivates Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA), offering a promising strategy for a chronic hepatitis B (CHB) cure. This method significantly outperforms APOBEC-mediated degradation of HBV DNA.
Area of Science:
- Virology
- Molecular Biology
- Gene Therapy
Background:
- Chronic Hepatitis B (CHB) remains incurable, with over 200 million infected globally.
- Hepatitis B virus (HBV) persists due to stable, nuclear covalently closed circular DNA (cccDNA).
- Current nucleoside analogue therapies inhibit viral DNA synthesis but do not eliminate cccDNA.
Purpose of the Study:
- To investigate the synergistic potential of CRISPR/Cas9 and cytokine-induced APOBEC mechanisms for cccDNA inactivation.
- To comprehensively analyze cccDNA mutations after Cas9 cleavage and repair via nonhomologous end joining (NHEJ).
Main Methods:
- CRISPR/Cas9 system application to cleave HBV cccDNA.
- Next-generation sequencing (NGS) for detailed mutation spectrum analysis of cccDNA.
- Comparison of CRISPR/Cas9 efficiency with APOBEC-mediated cytosine deamination induced by interferon-alpha (IFNα).
Main Results:
- CRISPR/Cas9 successfully cleaved over 90% of HBV DNA.
- CRISPR/Cas9 editing of HBV DNA was over 15,000 times more efficient than APOBEC-mediated deamination.
- The 3D-PCR method was found to overestimate edited HBV DNA frequency.
Conclusions:
- The CRISPR/Cas9 system is the most effective method to date for functionally inactivating HBV cccDNA.
- CRISPR/Cas9 holds significant promise as a curative therapy for chronic hepatitis B.
- Further research into gene-editing strategies is crucial for developing a cure for CHB.
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