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bioRxiv · 10.64898/2026.10.29.762891

Marked Mutants Allow for Rapid Non-Invasive Genotyping of Zebrafish by Visual Inspection Alone

Keatinge, M.·constantinou juhasz, S.·Shaw-McGrath, T.
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Abstract

Genetic analysis in zebrafish relies heavily on invasive, labour-intensive molecular genotyping. This limits throughput and phenotypic analysis, particularly for homozygous mutants that lack readily detectable phenotypes at early developmental stages. To overcome these limitations, we adapted CRISPR-mediated knock-in strategies to insert complete secondary marker cassettes (including promoter, fluorophore and polyA), directly into coding exons of target loci. This simultaneously disrupts target gene function and permanently labels mutant alleles, which we term Marked Mutants. Crossing heterozygous Marked Mutant alleles targeting the same gene but carrying distinct fluorescent secondary markers linked to each allele, enables unambiguous non-invasive visual identification of wild-type, heterozygous and biallelic mutant progeny as early as 24 hours post fertilisation. To test the scalability and robustness of our approach, we generated eight independent Marked Mutant alleles targeting mitfa, tdp43 and tdpl, using secondary markers comprising hatching gland (he1.1), pancreas (ins) or pineal gland (exorh) promoters driving expression of GFP, RFP or BFP. We found germline transmission efficiencies comparable to conventional CRISPR mutagenesis, with approximately 30% of fluorescence-selected F0 adults transmitted on-target insertions to the F1 generation. All Marked Mutant alleles behaved as loss-of-function alleles, demonstrating effective disruption of target gene function. By combining four independent Marked Mutant alleles, each carrying a distinct fluorescent secondary marker, we could prospectively identify tdp43/tdpl double compound heterozygous embryos by visual inspection alone before phenotypic onset. Only embryos carrying all four Marked Mutant alleles developed the characteristic lethal double-knockout phenotype, while siblings carrying fewer alleles remained phenotypically normal. By eliminating routine PCR genotyping and invasive fin clipping, Marked Mutants simplify colony management, facilitate combinatorial genetics, reduce animal use, and provide a practical platform for rapid non-invasive zebrafish reverse genetics.

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Keatinge, M., constantinou juhasz, S., Shaw-McGrath, T.. 2026-11-03. Marked Mutants Allow for Rapid Non-Invasive Genotyping of Zebrafish by Visual Inspection Alone. https://doi.org/10.64898/2026.10.29.762891

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