Engineered Allosteric Control Enhances Specificity and Potency of CRISPR-CasX-based Epigenetic Repressors

CRISPR-based epigenetic editing enables durable and reversible control of gene expression, overcoming the transient nature of RNA interference and antisense oligonucleotides while avoiding the permanent DNA alterations of other CRISPR-based approaches. Current epigenome editors that fuse the DNMT3A catalytic domain to a DNA-binding module such as catalytically inactive Cas9 deposit CpG methylation to silence gene transcription, but can exhibit off-target activity, cellular toxicity, and transcriptome-wide perturbations due to unconstrained methyltransferase activity. In contrast, DNMT3A regulation in its native context involves autoinhibition of its catalytic activity by its ATRX-DNMT3A-DNMT3L (ADD) domain, which is relieved only upon allosteric activation through recognition of unmodified H3K4 (H3K4me0). Here, we engineered this endogenous allosteric control into CRISPR-based epigenetic editors by designing compact Epigenetic Long Term CasX-based Repressors (ELXRs) containing an allosterically-gated DNMT3A. Allosteric ELXR substantially reduced off-target methylation, rescued dose-dependent growth defects in methylation-sensitive systems, and maintained or enhanced on-target activity across multiple loci (on average [≥]4-fold increased activity). We find that integration of DNMT3A allostery is more effective within the CasX framework than with Cas9-based systems, indicating that the increased precision targeting of CasX may be required for the allosteric gate to reduce off-target effects. Mechanistically, the engineered allostery establishes a multi-step gating process in which DNMT3A activation and subsequent CpG methylation occur only after conversion of H3K4me3 to H3K4me0, which we establish requires potentiation by a transcriptional repressor domain. Targeted mutagenesis of ADD confirms this allosteric mechanism in ELXRs matches endogenous DNMT3A regulation. Transcriptome-wide profiling further demonstrated substantially improved specificity with allosteric ELXRs, which were found to reduce the number of differentially expressed genes by 10-100-fold compared with their non-allosteric counterparts. In vivo, lipid nanoparticle delivery of ELXRs achieved potent, durable PCSK9 silencing with precise on-target promoter methylation and minimal off-target transcriptional effects. To date, CRISPR specificity has been governed primarily by gRNA-DNA interactions. By synthetically reconstructing DNMT3As allosteric control, we introduce a new regulatory layer that enhances the fidelity and specificity of CRISPR-based epigenetic editing and establishes a general framework for engineering more precise CRISPR-based therapeutics.