The opening keynote set an ambitious tone. Wendy Bickmore, director of the MRC Human Genetics Unit at the University of Edinburgh and a pioneer in functional three-dimensional genome organization, delivered the special EMBO Keynote lecture on how the non-coding genome regulates gene activity. She illustrated that distant enhancers do not always need direct physical contact with their target promoters to activate gene expression, but they must be located within roughly 200 to 300 nanometers of a promoter to function effectively. Bickmore showed that cohesin-mediated loop-extrusion is essential for long-range enhancer action, while also challenging the loop-extrusion model as the sole explanation, presenting evidence from her laboratory of ‘leaky’ insulation across topologically associating domain boundaries that causes bystander activation. She also unveiled a novel histone post-translational modification, H3K115 acetylation, which sits within the histone core rather than on the tail and is enriched precisely at the transcription start site in the nucleosome-depleted region, suggesting it marks a fragile nucleosome strongly associated with active transcription.

On the second day, Lei Stanley Qi, associate professor of bioengineering at Stanford University and a pioneer in CRISPR technology, offered a conceptual reframing of how scientists should interpret chromatin maps. TAD boundaries, he argued, are merely snapshots of chromatin interactions, whereas a cell functions as a living, dynamic system. He drew an evocative parallel with Leonardo da Vinci’s Vitruvian Man, which captures optional configurations of the human body, much as epigenetic figures capture the states of many cells across time and space. Qi described an epistasis mapping approach to enhancer functionality, explaining how redundant, independent, or synergistic enhancer interactions can provide compensatory, fine-tuning, or robust regulatory effects respectively, and noted that BRD4-mediated condensation can facilitate such interactions. Notably, his findings independently confirmed Bickmore’s conclusion that enhancers need spatial proximity to promoters to exert their effects.

The final keynote, from Angelo Lombardo, professor of tissue biology and regenerative medicine at Vita-Salute San Raffaele University and co-founder of Chroma Medicine, delivered perhaps the strongest evidence yet that epigenetic editing can work as a durable therapy. His laboratory discovered that stable gene repression requires simultaneous methylation of DNA and repressive histone marks, and that epigenetic regulators such as DNMT3A/3L and KRAB domains, which catalyze DNA and histone methylation, silence retroviral elements in embryonic stem cells. Inspired by this natural system, the group fused DNMT3A/3L and the KRAB domain of ZNF10 to zinc finger domains targeting specific genomic regions, achieving stable gene repression across multiple cell lines. They recently demonstrated long-term repression of PCSK9, a hepatocyte gene involved in cholesterol homeostasis, in mice for nearly one year, reducing LDL receptor presence on hepatocyte membranes. Strikingly, even after partial liver resection, the induced DNA methylation state and repressed expression were retained in the regrown tissue, showing that these modifications are faithfully inherited. Lombardo also highlighted epigenetic silencing of Hepatitis B virus via induced DNA methylation as a viable therapeutic avenue.

Beyond the keynotes, a wave of technical innovation is reshaping how epigenetic enzymes are understood and improved. Albert Jeltsch of the University of Stuttgart uses Deep Enzymology to systematically investigate the preferred flanking DNA sequences of DNA methyltransferases, finding that different DNA substrates influence DNMT1 efficacy so drastically that allele-specific DNA methylation becomes possible. Saulius Klimašauskas and colleagues at Vilnius University developed a click-chemistry technique for bioorthogonal labeling at sites where individual DNMTs catalyze methylation in live cells, enabling high-resolution chemical ‘tracks’ of epigenetic writers throughout the cell cycle. At Rice University, Jacob Goell reduced the cytotoxicity of the histone acetyltransferase P300 while preserving its enzymatic activity through a single point mutation in the P300 core, and found that P300 primes genes for activation and enhances prime editing efficiency independently of its catalytic function.

Epigenetic reader domains, which evolved over millions of years to recognize specific post-translational modifications, are also becoming central tools, particularly where high-quality antibodies are hard to develop. Tuncay Baubec of Utrecht University presented ChromID, a systematic approach in which a biotin ligase fused to a chromatin reader domain biotinylates nearby proteins; after pull-down and mass spectrometry, the epigenetic proteome at a targeted modification is mapped. EpiCypher’s Matthew Meiners described chimeric tandem reader domains and synthetic, fully chemically defined modified nucleosomes for use as spike-ins in CUT&RUN and CUT&Tag assays, allowing researchers to map reader-PTM interactions and histone modification co-occurrences. Anja Köhler of the University of Stuttgart introduced the Bimolecular Anchor Detector, or BiAD, technology, which combines a sgRNA/dCas9 DNA-binding module with reader-domain detector modules fused to complementary split fluorophore parts, reconstituting fluorescence only when both modules bind in proximity, thereby visualizing epigenetic marks at specific genomic loci in living cells. Meanwhile, Marvin Tanenbaum of the Hubrecht Institute presented stopless-ORF circular mRNAs encoding SunTag epitopes, permitting live-cell single-molecule imaging of ribosome kinetics and revealing that ribosome collisions actually facilitate translation through difficult sequences by resolving stalls.

As the toolbox expands, so does the appetite for unbiased, high-throughput screening. Lacramioara Bintu’s laboratory at Stanford developed dCas9-mediated high-throughput recruitment, or HT-recruit, testing more than 5,000 nuclear protein Pfam domains of human and viral origin for their ability to silence or activate gene expression, alongside a library of 114,288 sequences tiling transcription factors and chromatin regulators. Her characterization was memorably vivid: transcriptional activators tend to be ‘greasy acidic noodles with a little salt, pepper and queso,’ rich in acidic and hydrophobic residues interspersed with serine, proline, or glutamine, while repressors come in more flavors, with KRAB domains the best performers across contexts, including a newly identified KRAB from ZNF705F that outperforms the commonly used ZNF10 KRAB. At UC Berkeley, Michael Herschl screened over 50,000 pairs of epigenetic editors, some with catalytic domains up to 6.3 kilobases, using the COMBINE inducible screening platform, identifying editor combinations that impart long-term epigenetic changes and a bidirectional CRISPR perturbation system capable of activating and repressing genes concurrently. A key insight: domains operating in the same or similar pathways show good perturbation synergy, mimicking their natural collaboration. Samuel Reisman of Duke University, in work toward regenerative therapies, screened over 1,600 human transcription factors with CRISPR activation followed by Perturb-seq single-cell RNA sequencing to map the fidelity and subtype-specificity of astrocyte-to-neuron reprogramming.

Computational advances featured prominently as well. Kim Kira Witetzek of Academia Sinica presented ATAC-Mass, which combines isotopic labeling, ion beam imaging at 100-nanometer resolution, and mass cytometry to integrate epigenomics, proteomics, and three-dimensional nuclear imaging at the single-cell level. Jennifer Spangle of Emory University School of Medicine described a chemoenzymatic technique using an L-methionine analogue that converts into a SAM analogue, tagging methylated proteins with a detectable alkyne; the approach resolves mono-, di-, and trimethylation, histidine methylation, and arginine methylation with site specificity, works in vivo and across the blood-brain barrier, and identified 221 proteins with novel methylation sites upon enrichment. Philipp Schnee of the University of Stuttgart showed how 3D molecular dynamics simulations can predict enzyme behavior, enabling the design of a ‘Super-Substrate’ for protein lysine methyltransferases that outcompetes natural substrates with substantially increased specificity. Kimberley Glass of Brigham and Women’s Hospital presented SPIDER, a computational modeling tool that builds gene regulatory networks from DNase-seq, ATAC-seq, or DNA methylation data, prunes false positives using chromatin states, and accurately predicts ChIP-seq transcription factor binding events lacking a corresponding sequence motif, a persistent weakness of older pipelines.

The field’s clinical momentum was matched by growing candor about its risks. Jamie Hackett of EMBL Rome used CRISPR-dCas9 perturbation screens to dissect causal regulatory roles, finding that blocking histone tail acetylation prevents transcriptional activation after H3K4me3 deposition, and that gene permissiveness to epigenetic reprogramming depends on cell type, expressed factors, and DNA sequence. Domitilla del Vecchio of MIT proposed that H3K9me3 causally follows DNA methylation, noting that KRAB alone does not confer long-term memory while DNMT3A does. Bas van Steensel of the Netherlands Cancer Institute used transposon systems to relocate enhancers, promoters, and CTCF sites across a two-megabase window, discovering that enhancers communicate with gene bodies as well as promoters. On safety, Henriette O’Geen of UC Davis showed that hundreds of CpGs retain off-target methylation 24 days after transient editing, particularly at bivalent genes poised for transcription and implicated in oncogenesis, prompting a call for a gold standard for reporting off-target effects; Majid Pahlevan Kakhki of Karolinska Institutet independently reported widespread unintended methylation across nearly all CRISPR-dCas9 tools, including CRISPRoff. In applied settings, Pernette Verschure’s group found transcription burst size predicts gene responsiveness in hormone-sensitive breast cancer, while Gabriella Ficz of Barts Cancer Institute demonstrated ex vivo epigenetic editing of CDKN2B in umbilical cord hematopoietic stem cells, with durable methylation maintained after engraftment in mice and inherited across myeloid and lymphoid lineages. Ivana Parker of the University of Florida, meanwhile, mapped the epigenetic pathways underlying BCG vaccine-induced macrophage activation. Together, the Amsterdam meeting captured a field in confident transition: from conceptual understanding of chromatin’s static architecture to the dynamic, engineered, and increasingly therapeutic manipulation of the epigenome, balanced by a maturing commitment to safety, standardization, and open collaboration between academia and industry.

Subject of Research: Epigenetic editing technologies and their translation from bioengineering innovations toward clinical therapies

Article Title: Bridging bioengineering and epigenetics: from technical innovations to clinical applications

Article References: Jacob, J., van Loosen, Q. C., van den Berg van Saparoea, A. C. H., Sarno, F., & Verschure, P. J. (2024). Bridging bioengineering and epigenetics: from technical innovations to clinical applications. Epigenetics Communications, 4(1), Article 8. https://doi.org/10.1186/s43682-024-00031-x

Image Credits: AI Generated

DOI: 10.1186/s43682-024-00031-x

Keywords: epigenetics, bioengineering, CRISPR, epigenome editing, DNA methylation, chromatin, gene regulation, clinical applications, off-target effects, high-throughput screening, transcription, gene therapy

The review begins where the technology itself began: in prokaryotes. CRISPR-Cas systems evolved as adaptive immune defenses in bacteria and archaea, storing fragments of viral DNA in genomic memory banks and using them to recognize and destroy invading genetic material. When researchers realized that this bacterial immune machinery could be reprogrammed with a single guide RNA to target nearly any sequence of interest, the implications were enormous. Early CRISPR-Cas9 experiments demonstrated remarkable efficiency and ease of use compared with earlier genome-editing platforms such as zinc-finger nucleases and transcription activator-like effector nucleases, which required laborious protein engineering for each new target.

Yet the honeymoon was short-lived. As clinical ambitions grew, so did concern over off-target effects, the unwanted edits that occur when the Cas9 nuclease acts at sites that only partially match the guide RNA. Variable editing outcomes, unpredictable insertions and deletions at the target site, and the risk of large genomic rearrangements all limited the technology’s broader application to human therapy. The review emphasizes that these technical shortcomings, alongside delivery inefficiencies that make it difficult to get editing machinery into the right cells in the right tissues, remain among the field’s most persistent challenges even as the toolkit has grown far more sophisticated.

The evolution from scissors to editors is best embodied by the rise of base editing and prime editing. Rather than relying on double-strand breaks and the cell’s error-prone repair pathways, these newer approaches chemically convert individual DNA letters or write short new sequences with minimal collateral damage. The review highlights work demonstrating that prime editing can correct oncogenic KRAS variants without the constraints imposed by traditional CRISPR-Cas9 workflows, and that CRISPR-based strategies have achieved efficient correction of both KRAS and TP53 mutations, two of the most frequently mutated genes in human cancer. Such results illustrate the conceptual shift: the goal is no longer simply to disrupt a gene but to rewrite it precisely, restoring normal function rather than merely silencing a faulty instruction.

Cancer therapy has emerged as one of the most active arenas for these refined tools. The review surveys how CRISPR-Cas9 library screening has accelerated anti-cancer drug discovery by systematically identifying genes that tumors depend on, and how ex vivo editing of immune cells is reshaping immunotherapy. Chimeric antigen receptor T cells engineered with CRISPR have shown that targeting the CAR construct to a defined genomic locus, such as the TRAC locus, can enhance tumor rejection compared with randomly integrated vectors. Editing of natural killer cells and T cells from human donors is being pursued for cancer immunotherapy, and strategies to improve CAR-T cell persistence through CRISPR-mediated knockout of exhaustion-related pathways are advancing through preclinical and clinical pipelines.

Beyond cancer, the review catalogs applications across viral disease, autoimmune conditions and inherited disorders. CRISPR-based approaches are being developed to engineer resistance to viruses, with HIV-1/AIDS therapy representing a prominent example in which editing aims to remove or disable viral genomes integrated into host DNA. Diagnostic platforms derived from CRISPR nucleases, including SHERLOCK and DETECTR, exploit the sequence-specific recognition of Cas enzymes to detect viral nucleic acids with high sensitivity, offering rapid tools for emerging infectious diseases. In autoimmune disease research, genome editing is being used to dissect and potentially correct the immune dysregulation that underlies conditions such as lupus and rheumatoid arthritis, pointing toward a future in which the immune system itself can be retuned at the genetic level.

None of these therapeutic visions can be realized without solving the delivery problem, and the review devotes substantial attention to the technologies that ferry editing machinery into cells. Viral vectors, particularly adeno-associated virus and lentiviral vectors, offer efficient gene transfer but raise questions about payload capacity, insertional risks and immune responses. Non-viral alternatives include physical methods such as electroporation and microinjection, lipid nanoparticles and solid lipid nanoparticles, cell-penetrating peptides, DNA nanocarriers and extracellular vesicles. Nanotechnology-based delivery of CRISPR components for cancer treatment is a particularly active frontier, with gold nanoparticles and other platforms being evaluated for biocompatibility and cytotoxicity under evolving regulatory frameworks. The choice of delivery vehicle, the review makes clear, often determines whether an elegant editing strategy can ever reach the clinic.

Even when editing succeeds, the human body may object. Immunogenicity of the bacterial Cas9 protein is a recognized concern, since many people carry pre-existing antibodies and T cells reactive to common Cas variants from environmental exposure to the microbes that harbor them. The review also confronts the ethical dimensions that shadow every advance in the field. The prospect of human germline and heritable genome editing has prompted a global policy debate about where the lines should be drawn, and ethicists have argued that the moral considerations raised by such a powerful tool demand attention to accessibility, consent and the long-term consequences of permanent genomic change. Who will be able to afford these therapies, and how societies will govern their use, remain unresolved questions that the technical community cannot answer alone.

What emerges from the review is a portrait of a technology on a clear trajectory. Engineered Cas variants, such as PAM-flexible chimeric Cas9 proteins that relax the sequence constraints on where editing can occur, are expanding the addressable space of the genome. High-fidelity nucleases reduce off-target activity, base and prime editors increase the precision of the intended change, and improved delivery systems broaden the range of treatable tissues. Together these refinements position CRISPR as an increasingly precise platform for both fundamental research and therapeutic use, supporting targeted therapies for genetic disorders in ways that the original scissor paradigm could never achieve. The authors, based at the University of Science and Culture and the Royan Institute in Tehran, frame this progression not as a completed project but as an ongoing evolution toward greater specificity, safety and predictability.

The clinical momentum is already visible. Approved CRISPR-based therapies for hemoglobin disorders have demonstrated that genome editing can deliver durable benefit in patients, and dozens of trials are testing edited cell therapies, in vivo editing and CRISPR-enabled diagnostics across a widening spectrum of disease. The remaining obstacles, off-target editing, delivery inefficiency, immune reactions and equitable access, are formidable but no longer conceptual; they are engineering and policy problems with active research programs attached to each. As Rahbari, Lohrasbi and Amiri-Yekta conclude, the story of therapeutic genome editing is one of steady refinement, in which the crude power of the original molecular scissors is being progressively replaced by the controlled, predictable hand of a true genome editor. The precision revolution they describe is redefining what medicine can attempt at the level of DNA itself, and the pace of that redefinition shows no sign of slowing.

Subject of Research: The evolution of CRISPR-based genome editing from early nuclease cutting tools toward precise therapeutic platforms for genetic disease

Article Title: From scissors to editors: how the evolution of precision is redefining therapeutic genome editing

Article References: Rahbari, M., Lohrasbi, R., & Amiri-Yekta, A. (2026). From scissors to editors: how the evolution of precision is redefining therapeutic genome editing. Molecular Genetics and Genomics, 301(1), Article 193. https://doi.org/10.1007/s00438-026-02467-3

Image Credits: AI Generated

DOI: 10.1007/s00438-026-02467-3

Keywords: CRISPR-Cas9, genome editing, gene therapy, base editing, prime editing, off-target effects, cancer immunotherapy, CAR-T cells, viral vectors, nanoparticle delivery, precision medicine, genetic disorders

Severe combined immunodeficiency is a group of inherited disorders characterized by impaired lymphocyte development, and mutations in more than twenty genes have been implicated in the condition, including CD3D, IL7R, IL2RG, RAG1, RAG2, PRKDC, LIG4, and DCLRE1C, the gene that encodes Artemis. Affected infants typically present with chronic diarrhea, oral thrush, skin rashes, and recurrent, refractory infections. Without early diagnosis and intervention, the disease can lead to life-threatening complications and a high risk of mortality. Since the 1960s, hematopoietic cell transplantation has been the primary treatment, and thousands of infants have been treated worldwide, but the Artemis-deficient form of the disease poses particular challenges that have driven researchers to seek alternatives.

Artemis is an endonuclease that resolves DNA hairpin intermediates during V(D)J recombination, the genome-scrambling process that generates the diverse repertoire of antigen receptors on lymphocytes. After the RAG1/2 recombinase complex cleaves recombination signal sequences, Artemis, activated by DNA-PKcs, nicks the closed DNA hairpin coding ends, creating a 3-prime overhang that primes subsequent junctional diversification. The enzyme also acts as a caretaker of genomic integrity through its participation in the non-homologous end-joining DNA repair pathway. When Artemis is absent, T and B cell development is arrested, producing a radiation-sensitive form of SCID. The clinical spectrum is highly heterogeneous: null mutations generally cause classical SCID with complete arrest of lymphocyte development, while hypomorphic alleles retaining partial nuclease activity can give rise to atypical phenotypes, including Omenn syndrome and leaky SCID.

The transplantation route itself is complicated by the very biology of the disease. Patients with radiosensitive SCID are systemically sensitive to alkylating agents, the standard myeloablative drugs used before transplantation to eradicate recipient hematopoietic stem cells. Clinical studies have also indicated that ART-SCID patients remain hypersensitive to ionizing radiation and suffer more infections long after receiving an allogeneic transplant. Even with an HLA-matched sibling donor, T and B cell reconstitution after transplantation is not as efficient in ART-SCID patients as it is in other SCID genotypes. Gene delivery strategies using lentiviral vectors carrying wild-type Artemis cDNA have been explored, and replacing a strong viral promoter with the endogenous human Artemis promoter achieved robust immune recovery in affected infants after in vivo transduction of hematopoietic stem cells. However, the lingering safety concern of insertional mutagenesis, in which semi-randomly integrating vectors may activate oncogenes, persists. Meanwhile, CRISPR-Cas9 mediated homology directed repair, another gene correction approach, remains difficult to apply clinically because quiescent hematopoietic stem cells are intrinsically resistant to HDR and the error-prone NHEJ pathway competes for repair, resulting in low efficiency.

Against this backdrop, base editors have emerged as a compelling alternative. Cytosine base editors employ cytidine deaminases such as rat APOBEC1 to convert C•G base pairs to T•A via a C-to-U intermediate, while adenine base editors use evolved adenine deaminases to convert A•T to G•C through A-to-I deamination. Both consist of an engineered deaminase fused to a Cas9-derived module for guide RNA-directed DNA targeting, and neither requires a donor DNA template or creates a double-strand break. Base editors have already entered clinical trials for sickle cell disease, beta-thalassemia, and familial hypercholesterolemia, and have been used to develop universal CAR T cells. In a striking precedent, researchers delivered a cytidine base editor by electroporation into human hematopoietic stem cells to correct the CD3D mutation that causes another form of SCID, achieving approximately 71.2 percent pathogenic-allele repair.

To systematically identify suitable editing targets for ART-SCID, the research team first compiled previously reported pathogenic point mutations in Artemis and mined the ClinVar database for additional disease-associated variants, filtering out 41 mutations documented as pathogenic and highly conserved throughout Artemis evolution. Using an established Artemis-knockout 293T cell line and a coding-joint reporter assay, an extrachromosomal V(D)J recombination system in which functional Artemis enables GFP expression quantified by flow cytometry, the team screened candidate variants. They newly identified four mutations, G6E, E10G, F19L, and L110R, that severely impair Artemis activity, reducing both its exonuclease activity toward single-stranded DNA and its endonuclease activity toward 3-prime overhang and DNA hairpin substrates. Structural modeling based on the wild-type Artemis crystal structure further revealed that mutations such as G118V, G135E, and L110R substantially reduce protein stability, and that G118 coordinates a conserved water molecule bridging residues critical for activity across the related SNM1 family members SNM1A and SNM1B.

From this catalog, the team selected three loss-of-function mutations amenable to correction by established base editors: c.49G>A (D17N) and c.404G>A (G135E), both targetable by adenine base editors, and c.181T>C (C61R), a representative cytidine-base-editor target. The clinical stakes of these variants are well documented. The c.49G>A mutation was identified in a Japanese SCID study in which two of eight patients harbored the homozygous variant, presenting before six months of age with severe infections and profound T cell deficiency. The c.404G>A mutation was reported in a study of four ART-SCID patients, two of whom carried the homozygous variant with a T-negative, B-negative, NK-positive immunophenotype; one presented with persistent varicella-zoster virus infection, and both ultimately succumbed to severe complications including multiorgan failure. Before editing, the team also assessed predicted bystander substitutions within the editing windows, finding that S62L moderately attenuates activity, I16M and Y133C cause minor reductions, T134A has negligible impact, and I16V has no detectable effect, confirming the three sites as suitable and comparatively safe targets.

The ex vivo editing results were encouraging. For the c.181T>C mutation, the team tested several cytidine deaminases incorporated into the BE4max backbone, including rat APOBEC1, APOBEC3A, and evoFERNY, alongside narrow-window variants and a combination of rat APOBEC1 with SpRY-HF1, a PAM-relaxed, high-fidelity Cas9 variant. Deep sequencing showed that rAPOBEC1-SpRY-HF1-BE4max paired with a suitable guide RNA achieved approximately 50 percent editing efficiency at the target site, with no significant off-target activity at any of five computationally predicted genomic loci per guide, as assessed by targeted deep sequencing and CRISPResso2 analysis. Flow cytometry confirmed that editing partially restored Artemis endonuclease activity in the mutant cells. Haplotype-level analysis of bystander mutations revealed that most co-occurring substitutions were silent due to codon degeneracy, with the predominant functional bystander being S62L, which only moderately reduces activity.

For the adenine base editor experiments, five editors were tested, including ABE7.10, ABEmax, ABE8e, ABE8e-SpRY-HF1, and ABE8e-NRRH. ABE8e achieved approximately 35 percent correction at the c.49G>A site, comparable to ABE8e-NRRH with a PAM-relaxed guide, and flow cytometry demonstrated significantly increased Artemis activity following correction, with no significant off-target mutations at predicted sites and a dominant bystander, I16V, that does not impair function. The c.404G>A site proved more difficult: ABE8e and ABE8e-SpRY-HF1 reached roughly 20 percent editing efficiency, but Artemis activity was not restored. The culprit appears to be a frequent bystander mutation, D136G, which completely abolishes enzyme activity. The authors suggest that prime editing, which couples Cas9 to a reverse transcriptase and an exogenous RNA template to enable precise nucleotide correction with markedly reduced bystander effects, may be better suited for mutations of this type.

The authors are candid about the limitations of the work. All editing experiments were performed in 293T cells, which provide a convenient and robust evaluation system but do not fully recapitulate the biology of human hematopoietic stem and progenitor cells, which exhibit distinct DNA repair pathway preferences and greater sensitivity to editing-associated cytotoxicity. Future studies in patient-derived CD34-positive cells will require clinically relevant delivery methods such as electroporation of ribonucleoprotein complexes, specialized culture conditions to preserve stemness, and long-term engraftment assays in immunodeficient mouse models to confirm safety and self-renewal capacity. The off-target analysis was also limited to predicted sites, and genome-wide evaluation will be needed. Moreover, base editing cannot address patients with homozygous large genomic deletions, though correcting a single allele in compound heterozygous patients is supported by the observation that parents carrying only one mutant allele remain asymptomatic. Nevertheless, by linking precise nucleotide correction to functional recovery of a critical immune gene, the study establishes a theoretical foundation for base-editing therapies for ART-SCID and related monogenic disorders, and underscores how rapidly evolving editor toolkits, including newer glycosylase-based editors capable of C-to-G and T-to-G conversions, continue to expand the range of mutations that may one day be corrected at the bedside.

Subject of Research: Base editing correction of Artemis gene mutations as a potential gene therapy approach for Artemis-deficient severe combined immunodeficiency

Article Title: Base editing of Artemis mutations ex vivo sheds light on gene therapy for Artemis-deficient SCID

Article References: Base editing of Artemis mutations ex vivo sheds light on gene therapy for Artemis-deficient SCID. (n.d.). https://doi.org/10.1007/s44307-026-00115-w

Image Credits: AI Generated

DOI: 10.1007/s44307-026-00115-w

Keywords: Artemis, ART-SCID, base editing, CRISPR, cytidine base editor, adenine base editor, V(D)J recombination, severe combined immunodeficiency, gene therapy, ex vivo editing, DCLRE1C, off-target effects

The scale of methylation disruption in cancer is staggering. Tumor genomes characteristically display widespread global hypomethylation, in which vast stretches of normally methylated DNA lose their marks, accompanied by focal hypermethylation concentrated at key regulatory regions, particularly the CpG-rich promoters of tumor suppressor genes. The result is an extensive rewiring of gene regulatory networks: genes that restrain cell division are switched off, repetitive elements that are normally locked down become active, and the carefully orchestrated patterns that define cell identity dissolve into chaos. These alterations are so consistent that methylation signatures now underpin diagnostic and prognostic assays used in clinics worldwide, helping pathologists classify tumors, predict patient outcomes, and even trace the tissue of origin of metastatic cancers.

Yet a profound gap separates correlation from causation. The overwhelming majority of cancer-associated methylation changes have never been proven to drive tumor behavior; they may instead be passive consequences of the transformed state. Distinguishing driver marks from passenger marks using observational methylome profiling alone has proved remarkably difficult, because the epigenome is dynamic and context-dependent. This ambiguity has real consequences for drug development. Conventional hypomethylating agents, such as nucleoside analogs that trap and deplete methyltransferase enzymes, can partially reverse cancer-associated methylation alterations and have demonstrated clinical efficacy in selected hematological malignancies. But these drugs act genome-wide, with no capacity to target a specific promoter or enhancer. Their blunt mechanism limits both mechanistic insight—since any observed effect could stem from thousands of altered loci—and therapeutic precision, since desirable and undesirable demethylation occur simultaneously across the genome.

CRISPR-based epigenome editing offers a fundamentally different approach. Rather than cutting DNA, these systems repurpose catalytically inactive Cas proteins, most famously dCas9, as programmable delivery vehicles. Guided to a chosen genomic address by a complementary guide RNA, dCas9 can be fused to methylation ‘writers’—enzymes such as DNMT3A and DNMT3L that install methyl marks—or methylation ‘erasers’, including TET1 catalytic domains and engineered demethylases that oxidize and remove them. Because the underlying DNA sequence is left untouched, the editing is, in principle, reversible, mirroring the reversible nature of the epigenetic alterations themselves. The result is a technology capable of quantitatively tuning methylation at individual promoters, enhancers, and CpG shores—transition zones flanking CpG islands that are frequent sites of cancer-associated methylation gain.

The experimental power of this approach lies in its precision. A single tumor suppressor promoter can be hypermethylated in a controlled setting to test whether silencing of that gene is sufficient to confer growth advantages, or demethylated to ask whether restoring its expression suppresses malignant behavior. Similar logic applies to enhancers, whose methylation status can govern the activity of oncogenes and immune-modulatory genes alike. By editing specific loci, researchers can interrogate mechanisms of chemoresistance, asking whether methylation changes at particular drug-response genes cause treatment failure or merely accompany it. They can also probe immune modulation, since methylation patterns influence the expression of antigen-presenting molecules and immune checkpoint pathways that determine whether tumors are recognized by the immune system. In this way, methylation editing transforms the epigenome from a readout of tumor state into an experimentally addressable variable.

The review also highlights the modular nature of modern CRISPR recruitment platforms, which extend the technology well beyond simple enzyme fusions. Systems such as SunTag, MS2-based scaffolds, and related multiplexing strategies allow multiple effector domains to be recruited to a single target site, amplifying the magnitude of methylation change or combining writers and erasers with transcriptional activators and repressors. Such platforms enable graduated, quantitative manipulation of methylation levels rather than all-or-nothing switching, which is critical because the relationship between methylation density and gene expression is often dose-dependent. Combined with single-cell and multi-omics readouts, these tools allow researchers to observe how a targeted methylation edit propagates through chromatin state, transcription, and ultimately cellular phenotype in diverse cancer models, from cell lines to patient-derived organoids.

None of this progress comes without caveats, and the authors are candid about the obstacles that stand between bench and bedside. Off-target activity remains a central concern: both the DNA-binding specificity of guide RNAs and the promiscuity of the tethered enzymes can produce unintended methylation changes at sites resembling the intended target, and even at sites tethered transiently by direct enzyme-to-dCas9 fusions. Because methylation changes can be heritable through cell division, a single off-target event could have lasting consequences. Context-dependency poses a further complication. The same edit can yield different outcomes depending on chromatin context, cell type, CpG density, and the developmental or disease state of the cell, complicating efforts to generalize results across models. Delivery and durability add clinical dimensions to these challenges: getting large editing constructs efficiently into tumor cells—whether in culture or in a patient—and ensuring that the methylation change persists long enough to be therapeutically meaningful remain unsolved engineering problems.

The path forward, the review argues, lies in integration. Locus-specific editing is poised to converge with comprehensive multi-omics profiling, in which methylome, transcriptome, chromatin accessibility, and three-dimensional genome architecture are measured simultaneously before and after an edit. Machine-learning-guided target selection promises to accelerate this process, mining large epigenomic datasets to prioritize the methylation changes most likely to act as drivers, and to predict how editing a given locus will ripple through regulatory networks. Rational combination therapies represent the clinical endpoint of this vision: methylation editing might, for instance, be paired with immune checkpoint inhibitors by demethylating and reactivating antigen presentation genes, or with conventional chemotherapy by erasing methylation marks that drive drug resistance. Each of these strategies depends on the causal knowledge that only targeted editing can generate.

The significance of this synthesis extends beyond the technical details. For a generation, epigenetic therapy has been constrained by an uncomfortable irony: doctors could alter the cancer epigenome, but only in a coarse, genome-wide fashion that revealed little about which alterations mattered. CRISPR-based methylation editing inverts that logic, making the epigenome writable at single-locus resolution and thereby testable in a way no previous technology allowed. If the challenges of specificity, delivery, and context can be met, the result would be a new class of therapeutics that silence oncogenes or resurrect tumor suppressors without touching a single base of DNA sequence. For now, the technology’s greatest value is as an engine of discovery—a way to finally separate the drivers from the passengers in the cancer methylome. But the trajectory is clear, and the authors’ message is that rewriting the cancer epigenome has moved from metaphor to laboratory reality, with clinical translation as the next horizon.

One reason methylation is such an attractive target for editing is its intrinsic stability. Unlike histone modifications, which turn over rapidly and often require continuous reinforcement, DNA methylation at CpG sites can be faithfully copied through cell division by maintenance methyltransferases that recognize hemimethylated DNA after replication. This means a single, well-placed edit has the potential to persist across many generations of cells, a property that is both a strength for durable therapy and a liability if the wrong loci are modified. The review’s emphasis on precision reflects this double-edged character of the methylation mark.

The choice of effector enzymes also matters greatly. DNMT3A, one of the writer domains commonly fused to dCas9, is itself a gene frequently mutated in hematological malignancies, illustrating how the machinery of methylation regulation is intimately entangled with cancer biology. On the eraser side, catalytic domains derived from the TET enzymes, which normally initiate active demethylation through oxidation of 5-methylcytosine, can be repurposed for targeted removal of marks. That these same pathways are dysregulated in tumors underscores why locus-specific manipulation is such a valuable experimental tool: it allows individual components of an altered network to be tested in isolation.

The work emerges from a research group with a sustained focus on cancer epigenomics at the University of Otago’s Department of Pathology and Molecular Medicine, and the article is published open access under a Creative Commons Attribution license, supported by New Zealand funding bodies including the Health Research Council and the Marsden Fund. Open availability is significant for a methods-oriented review, since widespread adoption of these editing approaches depends on researchers across disciplines and resource settings being able to evaluate the technical trade-offs in detail.

It is also worth noting that the review appears at a moment when epigenome editing more broadly is maturing beyond proof-of-principle demonstrations. The same dCas9 recruitment logic underlies efforts to edit histone marks, chromatin architecture, and transcriptional states, suggesting that methylation editing will ultimately function as one module within a larger toolkit for writing and rewriting the regulatory layer of the genome.

Subject of Research: CRISPR-based DNA methylation editing technologies for interrogating and potentially treating aberrant cancer epigenomes

Article Title: Rewriting the cancer epigenome: CRISPR technologies for DNA methylation editing

Article References: Sadeghi, N., Rodger, E. J., & Chatterjee, A. (2026). Rewriting the cancer epigenome: CRISPR technologies for DNA methylation editing. Cellular and Molecular Life Sciences. https://doi.org/10.1007/s00018-026-06429-1

Image Credits: AI Generated

DOI: 10.1007/s00018-026-06429-1

Keywords: cancer, epigenetics, DNA methylation, CRISPR, DNA methylation editing, dCas9, epigenome editing, tumor suppressor genes, off-target effects, epigenetic therapy, gene regulation, precision medicine