Transient and tunable CRISPRa regulation of APOBEC/AID genes for targeting hepatitis B virus

Kostyushev, D.; Brezgin, S.; Kostyusheva, A.; Ponomareva, N.; Bayurova, E.; Zakirova, N.; Kondrashova, A.; Goptar, I.; Nikiforova, A.; Sudina, A.; Babin, Y.; Gordeychuk, I.; Lukashev, A.; Zamyatnin, A.A.; Ivanov, A.; Chulanov, V.

Авторы: Kostyushev Dmitry, Brezgin Sergey, Kostyusheva Anastasiya, Ponomareva Natalia, Bayurova Ekaterina, Zakirova Natalia, Kondrashova Alla, Goptar Irina, Nikiforova Anastasiya, Sudina Anna, Babin Yurii, Gordeychuk Ilya, Lukashev Alexander, Zamyatnin Andrey A., Ivanov Alexander, Chulanov Vladimir
Журнал: Molecular therapy. Nucleic acids
Том: 32
Год издания: 2023
Издательство: Nature Publishing Group
Местоположение издательства: United Kingdom
Первая страница: 478
Последняя страница: 493
DOI: 10.1016/j.omtn.2023.04.016
Аннотация: APOBEC/AID cytidine deaminases play an important role in innate immunity and antiviral defenses and were shown to suppress hepatitis B virus (HBV) replication by deaminating and destroying the major form of HBV genome, covalently closed circular DNA (cccDNA), without toxicity to the infected cells. However, developing anti-HBV therapeutics based on APOBEC/AID is complicated by the lack of tools for activating and controlling their expression. Here, we developed a CRISPR-activation-based approach (CRISPRa) to induce APOBEC/AID transient overexpression (>4–800,000-fold increase in mRNA levels). Using this new strategy, we were able to control APOBEC/AID expression and monitor their effects on HBV replication, mutation, and cellular toxicity. CRISPRa prominently reduced HBV replication (∼90%–99% decline of viral intermediates), deaminated and destroyed cccDNA, but induced mutagenesis in cancer-related genes. By coupling CRISPRa with attenuated sgRNA technology, we demonstrate that APOBEC/AID activation can be precisely controlled, eliminating off-site mutagenesis in virus-containing cells while preserving prominent antiviral activity. This study untangles the differences in the effects of physiologically expressed APOBEC/AID on HBV replication and cellular genome, provides insights into the molecular mechanisms of HBV cccDNA mutagenesis, repair, and degradation, and, finally, presents a strategy for a tunable control of APOBEC/AID expression and for suppressing HBV replication without toxicity.
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Transient and tunable CRISPRa regulation of APOBEC/AID genes for targeting hepatitis B virusстатья

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