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Orthologous CRISPR/Cas9 systems for specific and efficient degradation of covalently closed circular DNA of hepatitis B virus. Cellular and molecular life sciencesстатья Исследовательская статья
Статья опубликована в высокорейтинговом журнале
Статья опубликована в журнале из списка Web of Science и/или Scopus- Авторы: Kostyushev D., Brezgin S., Kostyusheva A., Zarifyan D., Goptar I., Chulanov V.
- Журнал: Cellular and Molecular Life Sciences
- Том: 76
- Номер: 9
- Год издания: 2019
- Издательство: Birkhauser Verlag
- Местоположение издательства: Switzerland
- Первая страница: 1779
- Последняя страница: 1794
- DOI: 10.1007/s00018-019-03021-8
- Аннотация: Covalently closed circular DNA (cccDNA) of hepatitis B virus (HBV) is the major cause of viral persistence and chronic hepatitis B. CRISPR/Cas9 nucleases can specifically target HBV cccDNA for decay, but off-target effects of nucleases in the human genome limit their clinical utility. CRISPR/Cas9 systems from four different species were co-expressed in cell lines with guide RNAs targeting conserved regions of the HBV genome. CRISPR/Cas9 systems from Streptococcus pyogenes (Sp) and Streptococcus thermophilus (St) targeting conserved regions of the HBV genome blocked HBV replication and, most importantly, resulted in degradation of over 90% of HBV cccDNA by 6 days post-transfection. Degradation of HBV cccDNA was impaired by inhibition of non-homologous end-joining pathway and resulted in an erroneous repair of HBV cccDNA. HBV cccDNA methylation also affected antiviral activity of CRISPR/Cas9. Single-nucleotide HBV genetic variants did not impact anti-HBV activity of St CRISPR/Cas9, suggesting its utility in targeting many HBV variants. However, two or more mismatches impaired or blocked CRISPR/Cas9 activity, indicating that host DNA will not likely be targeted. Deep sequencing revealed that Sp CRISPR/Cas9 induced off-target mutagenesis, whereas St CRISPR/Cas9 had no effect on the host genome. St CRISPR/Cas9 system represents the safest system with high anti-HBV activity.
- Добавил в систему: Костюшев Дмитрий Сергеевич