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 promise for therapeutic applications. However, the large size of dCas proteins substantially impedes delivery using adeno-associated virus (AAV) vectors. Here, through iterative bioinformatics analysis, structure-guided predictions and functional assays, we identified and characterized a miniature subtype V-M CRISPR-Cas12m from Pelomicrobium methylotrophicum. PmCas12m exhibited flexible 5'-YTN-3' PAM-dependent recognition and robust double-stranded DNA-binding properties while lacking DNA cleavage activity, thus rendering it a valuable tool for epigenome editing. Cryo-electron microscopy structures of PmCas12m unveiled its molecular mechanism of target DNA binding. Guided by these structural insights, we used deep mutational scanning 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 infection in a mouse model. Collectively, these findings establish xCas12m as a versatile epigenome-editing platform with notable potential for treating diseases, paving the way for clinical translation of epigenetic therapies.