Minimal Cas9 ancestor enables durable epigenetic control

Feng Zhang and colleagues report that the engineering of a compact RNA-guided nuclease, NovaIscB, derived from the OMEGA system enables efficient and specific epigenome editing in mammalian cells. Through evolution-guided protein design and structural RNA optimisation, NovaIscB supports in vivo applications via single-vector delivery.

By: Gorm Palmgren - May. 8, 2025

The study addresses longstanding limitations in genome editors by enhancing the activity and specificity of the IscB enzyme, a minimal ancestor of Cas9, while maintaining a compact size suitable for adeno-associated virus (AAV) packaging. The research team from the Broad Institute of MIT and Harvard screened diverse IscB orthologs and used structure-guided domain insertions, rational mutagenesis, and loop recombination to increase guide RNA recognition and reduce off-target effects.

NovaIscB (614 aa) derived by evolution-guided protein engineering of wt OrufIscB (499 aa). Modified... — NovaIscB (614 aa) derived by evolution-guided protein engineering of wt OrufIscB (499 aa). Modified under the Creative Commons license from Figure 2a in Kannan et al. (2025) Nature Biotechnology, doi.org/10.1038/s41587-025-02655-3

NovaIscB achieved a ~100-fold activity improvement over the original OgeuIscB, with a shift in effective guide length from ~13 to ~16–20 nucleotides, enhancing targeting specificity. Structural analysis via cryo-EM revealed extended duplex recognition and unique catalytic features distinct from Cas9, such as a tripartite histidine cluster coordinating a single Mg²⁺ ion in the HNH domain.

The NovaIscB system was fused with a DNA methyltransferase and KRAB domain to generate OMEGAoff, a transcriptional repressor capable of stable gene silencing. In both cultured cells and mouse models, OMEGAoff delivered durable repression of genes, such as Pcsk9, with reductions in protein levels and serum cholesterol. A truncated ωRNA scaffold further facilitated AAV packaging and increased expression efficiency, enabling robust in vivo delivery.

Feng Zhang led this work at the Broad Institute of MIT and Harvard, and it was published in Nature Biotechnology on 7 May 2025.

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ArticleCMN BriefsNewsEpigenome editing (e-GE)