Structure-guided discovery and engineering of miniature CRISPR-Cas12m for epigenome editing

CRISPR-based epigenome editing represents a programmable strategy to precisely modulate gene expression, holding immense promise for therapeutic applications. However, the large size of the dCas proteins substantially impedes the delivery via adeno-associated virus (AAV) vectors. Here, through iterative bioinformatics analysis, structure-guided predictions, and functional assays, we identified and characterized PmCas12m, a novel miniature subtype V-M CRISPR-Cas12m. PmCas12m exhibited flexible 5’-YTN-3’ PAM-dependent recognition and robust double-stranded DNA binding properties, while lacking DNA cleavage activity, thus positioning it as an ideal tool for epigenome editing. Cryogenic electron microscopy (cryo-EM) structures of PmCas12m unveiled its unique molecular mechanism of DNA binding facilitating interference. Guided by these structural insights, we employed deep mutational scanning (DMS) and protein engineering to develop xCas12m, a hypercompact variant with highly potent and specific epigenome editing capabilities in human cells. We further constructed the xCas12m-CRISPRoff platform in a single AAV vector, which achieved durable epigenetic silencing and effective inhibition of hepatitis B virus (HBV) infection in a mouse model. Collectively, these findings establish xCas12m as a versatile epigenome editing platform with transformative potential for treating diseases, paving the way for clinical translation of epigenetic therapies.