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bioRxiv · 10.64898/2026.01.16.699911

Marker-based knockout genotyping in diploid model organisms

Kraemer, F.·Ratke, J.·Strobl, F.
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Abstract

Analysis of gene function in diploid organisms typically relies on molecular methods to distinguish wild-type, mono-allelic knockout, and bi-allelic knockout individuals, creating a major practical bottleneck for large-scale developmental studies. Here, we establish a modular visual genotyping approach that enables organism-level discrimination of knockout zygosity by tagging alternative disrupted alleles with spectrally distinct fluorescent markers. Implemented in the red flour beetle Tribolium castaneum, a two-marker strategy permits reliable identification of bi-allelic knockouts within mixed cohorts in a semi-random insertional mutagenesis framework. Further, a four-marker strategy using a targeted CRISPR/Cas9-based gene editing approach enables systematic generation and unambiguous recognition of individuals homozygous for both a disrupted gene of choice and a fluorescent reporter transgene. This design substantially reduces the workload associated with routine molecular genotyping while enabling genotype-resolved long-term live imaging of embryonic morphogenesis. Application to the extra-embryonic specification factor zerknullt 1 reveals haplosufficiency and a semi-lethal phenotype associated with variable morphogenetic outcomes. Together, our approach provides a scalable framework for functional analysis of essential developmental genes. Summary StatementVisual marker-based genotyping enables organism-level discrimination of knockout zygosity, reducing reliance on molecular assays and facilitating scalable, genotype-resolved live imaging of developmental gene function in diploid model organisms.

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Kraemer, F., Ratke, J., Strobl, F.. 2026-01-17. Marker-based knockout genotyping in diploid model organisms. https://doi.org/10.64898/2026.01.16.699911

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