CRISPR activation of A3С, A3D, and A3H suppresses HBV replication but does not affect cccDNA or the host genome.
As we showed, short-term activation of A3A and A3B effectively suppresses HBV replication but causes off-target deamination of the host genome. Therefore, we decided to analyze the antiviral efficacy of other members of the APOBEC3/AID family with previously reported anti-HBV activity (A3C, A3D, and A3H). We designed sgRNAs targeting promoters of the selected genes (Figure 6A) and analyzed the effects of CRISPRa of these genes in the HepG2 rcccDNA co-transfection model. Transcription ofA3C , A3D , and A3H was increased 2–20-fold (Figure 6B). HBV life cycle analysis revealed a 50% reduction in both pgRNA and cccDNA levels following CRISPRa, while HBsAg level remained unchanged (Figure 6C).