Abstract
Cytosine base editing achieves C•G-to-T•A substitutions and can convert four codons (CAA/CAG/CGA/TGG) into STOP-codons (induction of STOP-codons, iSTOP) to knock out genes with reduced mosaicism. iSTOP enables direct phenotyping in founders’ somatic cells, but it remains unknown whether this works in founders’ germ cells so as to rapidly reveal novel genes for fertility. Here, we initially establish that iSTOP in mouse zygotes enables functional characterization of known genes in founders’ germ cells: Cfap43-iSTOP male founders manifest expected sperm features resembling human “multiple morphological abnormalities of the flagella” syndrome (i.e., MMAF-like features), while oocytes of Zp3-iSTOP female founders have no zona pellucida. We further illustrate iSTOP’s utility for dissecting the functions of unknown genes with Ccdc183, observing MMAF-like features and male infertility in Ccdc183-iSTOP founders, phenotypes concordant with those of Ccdc183-KO offspring. We ultimately establish that CCDC183 is essential for sperm morphogenesis through regulating the assembly of outer dynein arms and participating in the intra-flagellar transport. Our study demonstrates iSTOP as an efficient tool for direct reproductive disease modeling and phenotyping in germ cells of the founder generation, and rapidly reveals the essentiality of Ccdc183 in fertility, thus providing a time-saving approach for validating genetic defects (like nonsense mutations) for human infertility.
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Acknowledgement
This work was supported by the National Key Research and Development Program of China (2021YFC2701400) and the National Natural Science Foundation of China (32000393, 32322017, 32288101). We would like to thank the Center of Cryo-Electron Microscopy (CCEM) at Zhejiang University and Li Wang (CCEM) for their technical support on TEM. We would like to thank Professor Qinghua Shi (University of Science and Technology of China) and Chunyu Liu (Fudan University) for providing the anti-DNAH17 antibody. We would like to thank the Imaging Core Facility of State Key Laboratory of Genetic Engineering and the Facility of IMIB (Institute of Metabolism and Integrative Biology) at Fudan University for technical support. We would like to thank Zhicheng Wu (Tongji University) for support on piRNA analysis. We would like to thank Professor Hua Diao (Shanghai Institute for Biomedical and Pharmaceutical Technologies) for technical support on CASA analysis.
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Gene-knockout by iSTOP enables rapid reproductive disease modeling and phenotyping in germ cells of the founder generation
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Wang, Y., Chen, J., Huang, X. et al. Gene-knockout by iSTOP enables rapid reproductive disease modeling and phenotyping in germ cells of the founder generation. Sci. China Life Sci. 67, 1035–1050 (2024). https://doi.org/10.1007/s11427-023-2408-2
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DOI: https://doi.org/10.1007/s11427-023-2408-2