A SPLIT NANOBODY-BASED CRISPR EPI-EDITING TOOLBOX FOR MODULAR, MULTIPLEXED IN VIVO NEURONAL GENE REGULATION

CRISPR-based epigenome editing has emerged as a powerful strategy to interrogate transcriptional regulation in the nervous system, yet its widespread use in vivo remains constrained by the size, rigidity, and limited versatility of conventional dCas9–effector fusions. Here, we introduce a split, nanobody-based CRISPR epi-editing toolbox specifically designed to expand the experimental flexibility of neuronal epigenome manipulation. The system decouples DNA targeting from effector function by fusing a nuclease-deficient dCas9 to a GFP-specific nanobody, while transcriptional activators, repressors, or chromatin-modifying enzymes are fused to GFP, enabling their modular and interchangeable recruitment in trans at defined genomic loci. This architecture substantially reduces construct size, improves compatibility with viral delivery, and allows rapid swapping or multiplexing of effector modules without redesigning the targeting component. We validate the toolbox across multiple experimental contexts, including neuronal cell lines, primary hippocampal neurons, and in vivo, using both gain- and loss-of-function paradigms. The platform enables promoter- and locus-specific regulation of neuronal genes, precise editing of histone acetylation and heterochromatin marks, and simultaneous control of multiple activity-dependent genes using multiplex guide RNA strategies. To further support in vivo applications, we generated a transgenic mouse line expressing the dCas9–nanobody selectively in neurons, enabling efficient viral delivery of effector modules and guide RNAs. This versatile split epi-editing system provides a flexible framework for dissecting promoter-specific regulation, gene redundancy and chromatin dynamics in the brain, and offers broad applicability for functional genomics, disease modeling, and target validation in neuroscience.