CRISPR/Cas9-Mediated Rapid Generation of Multiple Mouse Lines Identified Ccdc63 as Essential for Spermiogenesis.
Spermatozoa are flagellated cells whose role in fertilization is dependent on their ability to move towards an oocyte. The structure of the sperm flagella is highly conserved across species, and much of what is known about this structure is derived from studies utilizing animal models. One group of proteins essential for the movement of the flagella are the dyneins. Using the advanced technology of CRISPR/Cas9 we have targeted three dynein group members; Dnaic1, Wdr63 and Ccdc63 in mice. All three of these genes are expressed strongly in the testis. We generated mice with amino acid substitutions in Dnaic1 to analyze two specific phosphorylation events at S124 and S127, and generated simple knockouts of Wdr63 and Ccdc63. We found that the targeted phosphorylation sites in Dnaic1 were not essential for male fertility. Similarly, Wdr63 was not essential for male fertility; however, Ccdc63 removal resulted in sterile male mice due to shortened flagella. This study demonstrates the versatility of the CRISPR/Cas9 system to generate animal models of a highly complex system by introducing point mutations and simple knockouts in a fast and efficient manner.
School of Environmental and Life Science, University of Newcastle, Callaghan, New South Wales 2308, Australia. samantha.a.young@uon.edu.au.|Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, Japan. samantha.a.young@uon.edu.au.
Samantha A M
SA
Young
Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, Japan. hmiya003@biken.osaka-u.ac.jp.
Haruhiko
H
Miyata
Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, Japan. yuhkohs@biken.osaka-u.ac.jp.
Yuhkoh
Y
Satouh
Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, Japan. hkato@biken.osaka-u.ac.jp.|Graduate School of Medicine, Osaka University, Suita, Osaka 565-0871, Japan. hkato@biken.osaka-u.ac.jp.
Hirotaka
H
Kato
Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, Japan. nozawak@biken.osaka-u.ac.jp.|Graduate School of Medicine, Osaka University, Suita, Osaka 565-0871, Japan. nozawak@biken.osaka-u.ac.jp.
Kaori
K
Nozawa
Immunology Frontier Research Center, Osaka University, Suita, Osaka 565-0871, Japan. isotani@biken.osaka-u.ac.jp.
Ayako
A
Isotani
School of Environmental and Life Science, University of Newcastle, Callaghan, New South Wales 2308, Australia. john.aitken@newcastle.edu.au.
R John
RJ
Aitken
School of Environmental and Life Science, University of Newcastle, Callaghan, New South Wales 2308, Australia. mark.baker@newcastle.edu.au.
Mark A
MA
Baker
Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, Japan. ikawa@biken.osaka-u.ac.jp.|Graduate School of Medicine, Osaka University, Suita, Osaka 565-0871, Japan. ikawa@biken.osaka-u.ac.jp.|Immunology Frontier Research Center, Osaka University, Suita, Osaka 565-0871, Japan. ikawa@biken.osaka-u.ac.jp.
Masahito
M
Ikawa
D000818:Animals; D064113:CRISPR-Cas Systems / Q000235:genetics*; D008297:Male; D051379:Mice; D013081:Sperm Motility / Q000235:genetics / Q000502:physiology; D013091:Spermatogenesis / Q000235:genetics / Q000502:physiology*
genome editing; sperm motility; spermatogenesis; targeted mutagenesis