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Male germ cells undergo complex developmental processes eventually producing spermatozoa through spermatogenesis, although the molecular mechanisms remain largely elusive. We have previously identified somatic cell nuclear transfer-reprogramming resistant genes (SRRGs) that are highly enriched for genes essential for spermatogenesis, although many of them remain uncharacterized in knockout (KO) mice. Here, we performed a CRISPR-based genetic screen using C57BL/6N mice for five uncharacterized SRRGs ( Cox8c ,  Cox7b2 ,  Tuba3a/3b ,  Faiml , and  Gm773 ), together with meiosis essential gene  Majin  as a control. RT-qPCR analysis of mouse adult tissues revealed that the five selected SRRGs were exclusively expressed in testis. Analysis of single-cell RNA-seq datasets of adult testis revealed stage-specific expression (pre-, mid-, or post-meiotic expression) in testicular germ cells. Examination of testis morphology, histology, and sperm functions in CRISPR-injected KO adult males revealed that  Cox7b2 ,  Gm773 , and  Tuba3a/3b  are required for the production of normal spermatozoa. Specifically,  Cox7b2  KO mice produced poorly motile infertile spermatozoa,  Gm773  KO mice produced motile spermatozoa with limited zona penetration abilities, and  Tuba3a/3b  KO mice completely lost germ cells at the early postnatal stages. Our genetic screen focusing on SRRGs efficiently identified critical genes for male germ cell development in mice, which also provides insights into human reproductive medicine.

CRISPR/Cas9-based genetic screen of SCNT-reprogramming resistant genes identifies critical genes for male germ cell development in mice