Reducing the expression of pathogenic proteins holds great promise for the therapy of a wide range of human diseases. Epigenetic editing approaches are favoured over DNA-editing technologies, which have been associated with off-target sequence edits. However, current CRISPR-based epigenetic editors are too large for delivery in adeno-associated virus (AAV) vectors, which are approved for human use. Neumann, Bertozzi et al. describe a novel, more compact, epigenetic editor termed CHARM (coupled histone tail for autoinhibition release of methyltransferase) that mediates long-term transcriptional silencing in human cell lines in vitro and in mice, with minimal toxicity and off-target activity.

Neumann, Bertozzi et al. used a previously described CRISPRoff strategy to silence PRNP transcription in HEK293T cells by DNA methylation. CRISPRoff comprises a catalytically dead Cas9 protein fused to the catalytically active methyltransferase domain of DNMT3A (D3A) and the C-terminal domain of the activating co-factor DNM3TL (D3L), together with a single-guide RNA targeting the transcription start site of PRNP. PRNP remains durably silenced for up to 6 months after transient transfection, owing to extensive methylation across the promoter region. However, the CRISPRoff construct exceeds the packaging capacity of an AAV vector for in vivo use, and the bacterial enzyme Cas9, which can be chronically expressed from episomal AAV genomes, is likely to become antigenic. In addition, the D3A domain, which bypasses the autoinhibitory mechanism of full-length DNMT3A, is cytotoxic when overexpressed.