Development of a phenotype screening platform and genetic disease modeling in Xenopus

Abstract

Forward genetic screens and phenotypic analysis are fundamental approaches for understanding gene function and modeling human diseases. However, current model organisms present significant limitations for high-throughput phenotypic screening. Mammalian models such as mice are constrained by small numbers of embryos per reproductive cycle, extended developmental periods, and internal fertilization that limits embryo accessibility. While zebrafish offer certain advantages, their evolutionary distance from humans reduces translational relevance. Cell culture systems, though well- suited for high-throughput approaches, lack the tissue and organ complexity necessary for studying whole-organism phenotypes. Xenopus laevis presents unique advantages that address these limitations: the ability to obtain hundreds of developmentally synchronized embryos simultaneously, external development allowing continuous monitoring of embryonic changes, closer evolutionary relationship to humans compared to zebrafish, and large embryo size facilitating experimental manipulation. However, technical challenges have limited its adoption for systematic genetic screening, including temperature sensitivity of genome editing tools, labor-intensive phenotype analysis, and lack of accessible platforms for high-throughput screening. This dissertation presents the development of a comprehensive phenotype screening platform for Xenopus laevis that overcomes these technical barriers. First, I developed XenoScan, a flatbed scanner-based phenomics platform integrated with deep learning algorithms for automated high- throughput phenotype analysis, capable of monitoring hundreds of embryos simultaneously over multiple days. Second, I validated the use of temperature-independent LbCas12a-Ultra nucleases for efficient genome editing in Xenopus laevis at standard culture temperatures (20-22 °C), achieving >80 % phenotypic penetrance compared to conventional tools. Finally, I established a disease model of Neurofibromatosis type 1 (NF1) using optimized CRISPR-Cas12a with Xenopus laevis, recapitulating key disease features including neurofibroma formation and cardiac defects, with successful pharmacological rescue using through FDA-approved drug. Together, these advances establish Xenopus laevis as a viable platform for phenomics-scale genetic screening and disease modeling. The integration of automated phenotype analysis, temperature- optimized genome editing, and validated disease modeling provides a powerful toolkit for investigating genotype-phenotype relationships in vertebrate development and advancing therapeutic development for genetic diseases.