Improving Gene Silencing with Next-Gen Engineered Epigenetic Editors

By Stuart P. Atkinson

Engineering Epigenetic Editors Takes Gene Silencing Platforms A Step Closer to Therapeutic Evaluations: A new study describes how engineering improvements to CRISPR-based epigenetic editors may bring gene-silencing platforms closer to therapeutic evaluation.

Engineering Epigenetic Editors with Lower Off-target DNA Methylation

Epigenetic editing performed by complexes formed from "dead" Cas (dCas) proteins fused to epigenetic effector domains can induce long-term alterations in gene expression without permanently altering the underlying DNA sequence. Epigenetic editors that aim to silence gene expression in cells - such as CRISPRoff (Nunez et al., Tremblay et al., and Xu et al.) - combine the deposition of repressive histone modifications with DNA methylation to establish a heterochromatin-like, repressed state (Mao et al.) that can be passed down to daughter cells (Greenberg & Bourc'his). These approaches currently employ only the catalytic methyltransferase domain of DNA methyltransferase 3A (DNMT3A), whose constitutive activity leads to off-target DNA methylation. Another functional domain of DNMT3A - the ADD (ATRX-DNMT3A-DNMT3L) regulatory domain - normally prevents such aberrant DNA methylation at undesired genomic loci (Guo et al. and Otani et al.) thanks to an ability to bind unmethylated histone H3 lysine 4 tails (H3K4me0), thereby avoiding active promoters typically marked by H3K4me3 and only targeting repressed regions. Could integrating this domain into epigenetic editors that silence gene expression reduce off-target effects and make these high-precision gene-silencing platforms a more palatable prospect for the treatment of monogenic human diseases?

Cartoon of the allosteric ADD-containing construct
Illustration of the new dCAS10/dCasX constructs featuring a H3K4me0-binding/H3K4me3-avoiding ADD domain

In their new bioRxiv preprint, researchers led by Jason D Fernandes (Scribe Therapeutics) now report the integration of novel ADD-containing DNMT3A variants into a precision epigenetic editing platform using the compact, highly specific CasX (Cas12e) platform (Charles et al.), which the lab had previously optimized for high potency and fidelity. Their findings, which include in vivo evaluations, now suggest that this exciting strategy can enhance the precision, specificity, and therapeutic potential of CRISPR-based epigenetic editors, potentially accelerating the clinical translation of similar platforms.

The brand new CUT&TAPS technique combines CUT&Tag/scCUT&Tag with DNA methylation analysis to enable simultaneous profiling of chromatin state and underlying DNA methylation patterns. In relation to this exciting study, the application of CUT&TAPS could assay on-target (H3K4me0) and off-target (H3K4me1/2/3) histone modifications and simultaneously assay corresponding DNA methylation levels to directly validate the impact of the all-important ADD domain. Of note, CUT&TAPS is compatible with single-cell platforms, enabling a deeper understanding of crosstalk between chromatin and DNA at the single-cell level. Check out all the details concerning our scCUT&Tag Kits, scCUT&Tag Services, and bulk CUT&Tag Services at the Epigenome Technologies website. For more information on CUT&TAPS please send us a message!

Epigenetic Long-Term CasX-based Repressors - Improving On-Target and Reducing Off-Target Epigenetic Editing

Barplot of relative demethylation of the novel construct to ADD-noncontaining versions
Incorporation of the ADD H3K4me0-binding (H3K4me3-avoiding) domain results in reduced off-target DNA methyltation at chromatin-sensitive domains such as GAPDH. From Figure 1 of Charles et al.

As part of their exciting new study, the authors engineered novel DNMT3A variants that contained the ADD domain, an endogenous control module that prevents aberrant DNA methylation, and then created precision epigenetic editors built on the compact, highly specific CasX (Cas12e) platform, previously optimized for high potency and fidelity. This approach led to the development of what the team called "Epigenetic Long Term CasX-based Repressors" (ELXRs), which displayed substantially reduced off-target methylation, rescued dose-dependent growth defects in methylation-sensitive systems, and maintained/enhanced on-target activity across multiple target loci while displaying low cytotoxicity. Of note, the CasX system provided overall better results than Cas9 in this context; therefore, the two novel features of this epigenome editing approach likely provide synergistic activity, with increased precision targeting and reduced off-target effects observed. The authors state that ELXRs simultaneously improve efficacy and specificity by stabilizing productive chromatin interactions at target loci.

Boxplot of inadvertantly repressed genes
The addition of the ADD-domain to the construct results in far less repression of off-target, chromatin-state-dependent genes, compared to the original dCAS construct lacking the ADD domain. From Figure 3 of Charles et al.

At the mechanistic level, DNMT3A activation and DNA methylation occurred only after the conversion of H3K4me3 to H3K4me0 via potentiation by the transcriptional repressor domain, which supports a mechanism whereby the methyltransferase domain remains autoinhibited (preventing off-target methylation) until chromatin cues engage the ADD domain to enable the precise activation of methyltransferase activity when at the correct locus. Subsequent transcriptomic profiling demonstrated substantially improved specificity when using ELXRs; this analysis revealed a 10- to 100-fold reduction in the number of differentially expressed off-target genes compared to dCas9-based CRISPRoff systems. Finally, the authors explored the in vivo potential of ELXRs via lipid nanoparticle-mediated delivery in a mouse model; excitingly, they reported durable, precise promoter DNA methylation and silencing of the target PCSK9 gene (a therapeutically relevant and clinically validated locus for treating hypercholesterolemia; Rosenson et al.) with minimal off-target transcriptional effects.

Volcano plot of treatment-induced mRNA changes
Side-by-side comparison of gene repression via genomic editing using original (ADD-) and new (ADD+) constructs show a significant reduction in off-target gene modulation both in vivo and in vitro. From Figure 5 of Charles et al.

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Towards the Development of Therapeutically Relevant Precision Epigenetic Silencing Platforms

Overall, the authors created a precision epigenetic silencing platform with multiple advantages over other similar platforms, thanks to the addition of the ADD regulatory domain of DNMT3A. The significantly increased on-target and decreased off-target efficiency observed, along with the successful therapeutic application of this approach in vivo, may provide a general framework for the development of yet more precise CRISPR-based therapeutics.

The CUT&TAPS technique combines CUT&Tag/scCUT&Tag with DNA methylation analysis to enable simultaneous profiling of chromatin state and underlying DNA methylation patterns. In relation to this exciting study, the application of CUT&TAPS could assay on-target (H3K4me0) and off-target (H3K4me1/2/3) histone modifications and simultaneously assay corresponding DNA methylation levels to directly validate the impact of the all-important ADD domain. Of note, CUT&TAPS is compatible with single-cell platforms, enabling a deeper understanding of crosstalk between chromatin and DNA at the single-cell level. Check out all the details concerning our scCUT&Tag Kits, scCUT&Tag Services, and bulk CUT&Tag Services at the Epigenome Technologies website. For more information on CUT&TAPS please send us a message!