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Abstract

Volume 24, Issue 3 (May 2022) 24, 266–272; 10.4103/aja.aja_63_21

CRISPR/Cas9-mediated genome editing reveals 12 testis-enriched genes dispensable for male fertility in mice

Yuki Oyama1,2, Haruhiko Miyata2, Keisuke Shimada2, Yoshitaka Fujihara2,3, Keizo Tokuhiro4, Thomas X Garcia5,6,7, Martin M Matzuk5,6, Masahito Ikawa1,2,8

1 Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka 565-0871, Japan
    2 Department of Experimental Genome Research, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, Japan
    3 Department of Bioscience and Genetics, National Cerebral and Cardiovascular Center, Suita, Osaka 564-8565, Japan
    4 Department of Genome Editing, Institute of Biomedical Science, Kansai Medical University, Hirakata, Osaka 573-1191, Japan
    5 Center for Drug Discovery, Baylor College of Medicine, Houston, TX 77030, USA
    6 Department of Pathology and Immunology, Baylor College of Medicine, Houston, TX 77030, USA
    7 Department of Biology and Biotechnology, University of Houston-Clear Lake, Houston, TX 77058, USA
    8 The Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo 108-8639, Japan

Correspondence: Dr. H Miyata (hmiya003@biken.osaka-u.ac.jp) or Dr. M Ikawa (ikawa@biken.osaka-u.ac.jp)

16-Jul-2021

Abstract

Gene expression analyses suggest that more than 1000–2000 genes are expressed predominantly in mouse and human testes. Although functional analyses of hundreds of these genes have been performed, there are still many testis-enriched genes whose functions remain unexplored. Analyzing gene function using knockout (KO) mice is a powerful tool to discern if the gene of interest is essential for sperm formation, function, and male fertility in vivo. In this study, we generated KO mice for 12 testis-enriched genes, 1700057G04Rik, 4921539E11Rik, 4930558C23Rik, Cby2, Ldhal6b, Rasef, Slc25a2, Slc25a41, Smim8, Smim9, Tmem210, and Tomm20l, using the clustered regularly interspaced short palindromic repeats /CRISPR-associated protein 9 (CRISPR/Cas9) system. We designed two gRNAs for each gene to excise almost all the protein-coding regions to ensure that the deletions in these genes result in a null mutation. Mating tests of KO mice reveal that these 12 genes are not essential for male fertility, at least when individually ablated, and not together with other potentially compensatory paralogous genes. Our results could prevent other laboratories from expending duplicative effort generating KO mice, for which no apparent phenotype exists.
    
    Keywords: CRISPR/Cas9; knockout mice; male infertility; spermatozoa; testis

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