Initially, our idea was to design a system using Cas proteins to suppress viral DNA replication and protein expression through the cooperative action of dual dCas complexes. Such systems offered high interaction specificity, and due to the absence of nuclease activity, they were considered safer for the host genome.
However, after consultations with experts, we identified several limitations of this approach. The main challenges included the large size of Cas9 proteins, which complicated packaging into viral vectors and delivery to hepatocytes, as well as the need to incorporate additional regulatory domains such as KRAB repressors or DNA methyltransferases into fusion proteins. These modifications increased construct size and complexity while raising concerns about potential off-target effects caused by extended guide RNA architectures.
In light of these limitations, we redirected our focus toward constructing stable RNA–RNA interactions between pairs of guide RNAs while preserving precise target specificity. This approach allowed the creation of cooperative complexes compatible with different Cas proteins and provided a modular framework for building dual-CRISPR systems.
We believed that this strategy would simplify and accelerate the selection of efficient RNA dimers while enabling the development of solutions suitable for a wide range of epigenetic editors. Furthermore, the same principle could be applied to engineer analogous repression systems for other chronic viral infections. Based on these insights, we proposed a novel RNA-mediated platform for forming stable dimeric CRISPR complexes, designed for the treatment of hepatitis B and potentially other viral diseases.
Our work was aimed at developing a safe, specific, and modular system for the suppression of hepatitis B virus (HBV) transcription through epigenetic regulation rather than direct DNA cleavage. Current genome-editing technologies, including CRISPR/Cas9, were highly efficient at cutting DNA, but their therapeutic potential remained limited by off-target activity, double-strand breaks (DSBs), and the risk of genomic instability in host cells.
In the context of chronic HBV infection, these issues were particularly relevant because cccDNA, the viral episome responsible for persistence, existed in the nuclei of hepatocytes as a stable, small circular DNA molecule. Irreversible DSBs in this structure could lead to unintended chromosomal integration events or hepatotoxic effects. Therefore, direct destruction of cccDNA using nuclease-active Cas proteins was not considered a safe therapeutic strategy.
To overcome these limitations, we designed an epigenetic silencing system based on dCas proteins guided by dual sgRNAs capable of forming RNA–RNA dimerization loops.
This architecture allowed us to target cccDNA at two closely spaced regions, promoting cooperative binding of dCas complexes without introducing DNA breaks. The resulting spatially coordinated occupancy enabled long-term transcriptional repression of HBV genes while maintaining the integrity of the host genome.
Our approach also addressed another limitation of classical single-gRNA designs — insufficient specificity. The dual-guide architecture improved targeting precision through cooperative assembly, reducing off-target probability by more than an order of magnitude in model systems.
We considered this system particularly suitable for therapeutic applications, as its modular RNA-based design allowed flexible adaptation for other episomal or persistent viral genomes. The use of catalytically inactive Cas proteins eliminated mutagenic risks, while RNA-mediated complex formation minimized immunogenicity by reducing overall protein size compared with protein–protein dimerization systems.
In a broader context, the system could be integrated into combination therapy protocols, where epigenetic repression of cccDNA would be complemented by existing nucleos(t)ide analogues (such as tenofovir or entecavir) or direct antiviral drugs (e.g., Sofosbuvir and Velpatasvir).
By coupling transcriptional silencing with pharmacological inhibition of viral replication, it would be possible to achieve a functional cure — long-term suppression of viral activity without the need for continuous therapy.
The practical implementation of our technology was envisioned as a multi-stage process, integrating molecular design, preclinical validation, and delivery optimization.
In summary, our implementation strategy represented a clear translational pathway — from conceptual design to therapeutic application — emphasizing biosafety, reversibility, and modularity.