Gene-knockout by iSTOP enables rapid reproductive disease modeling and phenotyping in germ cells of the founder generation  

作　　者：Yaling Wang Jingwen Chen Xueying Huang Bangguo Wu Peng Dai Feng Zhang Jinsong Li Lingbo Wang 

机构地区：[1]State Key Laboratory of Genetic Engineering,Shanghai Key Laboratory of Metabolic Remodeling and Health,Institute of Metabolism and Integrative Biology,Fudan University,Shanghai 200438,China [2]Institute of Reproduction and Development,Obstetrics and Gynecology Hospital,Fudan University,Shanghai 200011,China [3]NHC Key Lab of Reproduction Regulation(Shanghai Institute for Biomedical and Pharmaceutical Technologies),School of Pharmacy,Fudan University,Shanghai 200433,China [4]Shanghai Key Laboratoryof Maternal and Fetal Medicine,Clinical and Translational Research Center of Shanghai First Maternity and Infant Hospital,Frontier Science Center for Stem Cell Research,Schoolof Life Sciences and Technology,Tongji University,Shanghai 200092,China [5]State Key Laboratory of Cell Biology,CAS Center for Excellence in Molecular Cell Science,Shanghai Institute of Biochemistry and Cell Biology,Chinese Academy of Sciences,Shanghai 200031,China

出　　处：《Science China(Life Sciences)》2024年第5期1035-1050,共16页中国科学（生命科学英文版）

基　　金：supported by the National Key Research and Development Program of China(2021YFC2701400);the National Natural Science Foundation of China(32000393,32322017,32288101)。

摘　　要：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.

关 键 词：disease modeling INFERTILITY multiple morphological abnormalities of the flagella(MMAF) induction of STOP-codons(iSTOP CRISPR-STOP) sperm motility 

分 类 号：Q78[生物学—分子生物学] R698.2[医药卫生—泌尿科学]