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

Marked Mutants enable non-invasive visual genotyping of zebrafish progeny as early as 24 hours post fertilisation, distinguishing wild-type, heterozygous, and biallelic mutants by fluorescence.

• Why it matters: Invasive PCR genotyping and fin clipping limit zebrafish throughput and complicate early-stage analysis of homozygous mutants lacking obvious phenotypes.
• What they did: The team used CRISPR-mediated knock-in to insert complete secondary marker cassettes into coding exons, then crossed heterozygous lines with distinct fluorescent markers to read genotypes visually.
• The result: In eight Marked Mutant alleles targeting mitfa, tdp43, and tdpl, about 30% of fluorescence-selected F0 adults transmitted on-target insertions to F1, and combining four alleles prospectively identified lethal double-knockout embryos before phenotypic onset.

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