Science Spotlight: C-ing Clearly
C-ing clearly: high-efficiency cytosine editors redefine disease-variant modelling
By Samhita Krothapalli
Often, a patient leaves a genetics clinic with a question instead of a diagnosis: their genome sequence has a mutation in a disease gene, but whether it is pathogenic or benign is unknown. When no clinician, database, or published literature can classify a variant in a disease-relevant gene, it earns an uncomfortable designation: “variant of uncertain significance”, or VUS. Now, Qin and colleagues in the Varshney lab at the Oklahoma Medical Research Foundation have engineered TadA, an adenine deaminase to recreate human disease variants in zebrafish and turn previously unresolved VUS into functional verdicts (Qin et al., 2026).
The road to this advance was paved by a decade of promising but imperfect base editors. Base editors introduce single-base conversions without generating the double-strand DNA breaks and consequent insertions and deletions that classical CRISPR does. Cytosine base editors (CBEs) that are built around APOBEC-family deaminases are the standard approach for C to T substitutions (Komor et al., 2016), but these can generate bystander edits at non-target cytosines within the editing window, and pervasive transcriptome-wide RNA off-target editing, limiting the very precision the field demands (Grünewald et al., 2019). TadA-derived cytosine editors offer a path forward, and the Varshney lab's own zTadCBE (Qin et al., 2025) demonstrated low indel rates and minimal sequence-context bias in zebrafish. However, on-target activity was less than optimal, failing to generate the levels of biallelic editing necessary to assess the effects of mutations directly in injected G0 fish. Improvements in CBEs were needed.
Qin and colleagues engineered a next-generation TadA-derived editor, called TCBE-Umax, with enhanced CBE efficiency. They engineered mutations at positions N46 and Y73 in the TadA deaminase domain that had been identified through phage-assisted evolution (Zhang et al. 2024), as well as V82S and Q154R mutations from the lab's own prior zebrafish work (Qin et al. 2025). Four candidate editors were screened across seven endogenous loci. TCBE-Umax, carrying all four substitutions, emerged as the top performer. Compared to zTadCBE, TCBE-Umax delivered twofold higher efficiency, with up to 89% editing, no detectable A-to-G activity, minimal bystander edits, and consistent performance regardless of sequence context. A family of derivative editors further expanded PAM flexibility, including near-PAMless NNN variants, and introduced precision-tuned editing windows. Together, these modifications address the targeting and specificity constraints of earlier CBEs.
Experimental workflow for VUS pathogenicity screening in F₀ zebrafish, from Qin et al. 2026
With a validated tool in hand, the authors turned to the biological question it was built to address. They focused on Usher syndrome, a hereditary hearing and vision loss disorder caused by mutations in MYO7A and CDH23. Qin et al. selected 15 missense variants from ClinVar: 14 VUS, and one likely pathogenic positive control. TCBE-Umax was injected with variant-specific guide RNAs into one-cell stage embryos, and injected larvae were assessed at 5 dpf for their acoustic startle response and sensory hair cell morphology. Affected larvae were genotyped to correlate phenotype with genotype (see figure). Amongst the 15 variants the group modelled, seven were conclusively classified as pathogenic and seven as benign, while only one remained ambiguous. This information, when communicated back to patient families with VUS diagnoses, will provide much-needed certainty about the genetic cause of their disease and their treatment path going forward.
The hearing loss screen serves as a proof of concept. Dr. Varshney says, “the platform is disease-agnostic — any gene with a zebrafish orthologue and a tractable phenotypic readout is potentially addressable”. With zebrafish orthologues in ~80% of human disease genes, this strategy could potentially open doors to conditions spanning cardiac, metabolic and neurodevelopmental disorders. More immediately, the framework addresses a key need in the clinical genetics community: a fast, organism-level route from sequencing report to functional verdict, at a scale that could meaningfully reduce the VUS backlog and hasten precise diagnoses.
However, no tool is perfect, and TCBE-Umax is no exception. Variants falling outside accessible editing windows, in repetitive genomic regions, or at loci prone to mosaic editing in F₀ embryos remain challenging, and editors optimised for zebrafish may need to be re-engineered for other model systems. What this paper establishes, unambiguously, is that the pipeline works: a VUS goes in, a functional verdict comes out, within days, in a living vertebrate, at a previously unprecedented scale. Improved delivery strategies and continued Cas9 engineering will likely sharpen this capability. Asked about the next steps, Dr. Varshney says: "The lab will keep pushing, until we have tools efficient and precise enough to model every single base in the zebrafish genome”, an ambition that provides hope for the millions of patients diagnosed with variants of unknown significance who are desperate for more information about the cause of their disease.
References:
Qin, W., Lin, SJ., Zhang, Y. et al. High-efficiency TadA cytosine base editors for precise modelling of human disease variants. Nat. Biomed. Eng (2026). https://doi.org/10.1038/s41551-025-01607-1
Porto, E. M. & Komor, A. C. In the business of base editors: evolution from bench to bedside. PLoS Biol. 21, e3002071 (2023). https://doi.org/10.1371/journal.pbio.3002071
Komor, A., Kim, Y., Packer, M. et al. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533, 420–424 (2016). https://doi.org/10.1038/nature17946
Grünewald, J., Zhou, R., Garcia, S.P. et al. Transcriptome-wide off-target RNA editing induced by CRISPR-guided DNA base editors. Nature 569, 433–437 (2019). https://doi.org/10.1038/s41586-019-1161-z
W.Qin, S.-J.Lin, Y.Zhang, et al. “Rationally Designed TadA-Derived Cytosine Editors Enable Context-Independent Zebrafish Genome Editing.” Adv. Sci.12, no. 39 (2025): e09800. https://doi.org/10.1002/advs.202509800
Zhang, E., Neugebauer, M.E., Krasnow, N.A. et al. Phage-assisted evolution of highly active cytosine base editors with enhanced selectivity and minimal sequence context preference. Nat Commun 15, 1697 (2024). https://doi.org/10.1038/s41467-024-45969-7
About the writer:
Samhita Krothapalli is completing her Master's in Molecular Bioengineering at TU Dresden, where zebrafish have become her model organism of choice, and at this point, something closer to colleagues. She is broadly interested in developmental biology and fascinated by the regenerative properties of zebrafish Müller glia and the translational potential of understanding these mechanisms for human retinal repair. Outside the lab, she enjoys hiking and exploring lesser-known places.
About the authors:
Gaurav K. Varshney, PhD, leads a research group in the Genes and Human Disease Research Program at the Oklahoma Medical Research Foundation in Oklahoma City. His laboratory develops scalable genome-editing technologies and functional genomics resources in zebrafish, with a particular focus on modelling rare human genetic diseases and resolving variants of uncertain significance. Recent work from the group includes ABE-Ultramax for high-efficiency adenine base editing and the TCBE-Umax family of cytosine base editors described here, both aimed at enabling rapid, organism-level functional assessment of disease-associated single-nucleotide variants.
Wei Qin, PhD, is a researcher in the Genes and Human Disease Research Program at the Oklahoma Medical Research Foundation in Oklahoma City. His research focuses on developing genome editing tools in zebrafish to model disease-associated variants and understand how different mutations in the same gene produce distinct disease phenotypes. Outside the lab, he enjoys traveling with his family and performing magic, a lifelong interest inspired by his grandfather, a professional magician.
Gaurav Varshney (left) and Wei Qin (right)