Organism-level visual genotyping of knockout zygosity enables functional gene analysis and modular combination with reporter transgenes

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 zerknüllt 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 Statement Visual 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.