Optimization of AsCas12f1-mediated long-term gene repression.

A compact and programmable epigenome editor can be utilized for long-term gene silencing, making it a powerful tool for biological research and biomedical applications. Here, we present an enhanced CRISPR-based epigenome editor based on deactivated Acidibacillus sulfuroxidans Cas12f1 (dAsCas12f1) variants, which incorporate epigenetic modulators for histone methylation and DNA methylation. First, we compared the gene silencing achieved by various dCas12f1 variants fused with Krüppel-associated box (KRAB). The optimized variant dCas12f1-V8.1-KRAB, named AsCas12f1-based miniaturized CRISPRi (AminiCRi), demonstrated high-efficacy gene silencing comparable to that of dSpCas9-KRAB. To establish a miniature Cas-based system for long-term silencing, we generated dCas12f1-V8.1 fused with KRAB and DNA methyltransferases (Dnmt3A/3L), named AsCas12f1-based miniaturized CRISPRoff (AminiCRoff). AminiCRoff enables simultaneous deposition of repressive histone H3K9 trimethylation marks and DNA methylation at target loci. Transient delivery of AminiCRoff allows stable suppression of H2B gene expression to 30% of baseline levels for up to 50 days, which is more durable than dSpCas9-based CRISPRoff. The compact size of the system is potentially packed into a single adeno-associated virus (AAV), which is frequently used for gene therapy. Here, we developed a highly efficient miniature Cas-based epigenetic editor for long-term gene silencing, which provides a good foundation for epigenetic therapy of genetic or chronic diseases with abnormal gene expression.