CRISPR-based genetic screen identified Cox7b2 and Gm773 as critical factors for the sperm fertilizing ability in mice

抄録

[Introduction] Spermatogenesis is a complex process involving meiosis and spermiogenesis, but still, the underlying molecular mechanisms are not fully understood. Here, we try to identify novel genes that play a critical role(s) in spermatogenesis by performing a CRISPR-based genetic screening in mice. [Materials and Methods] We selected five uncharacterized candidate genes (Cox7b2, Cox8c, Faiml, Gm773, and Tuba3b) and one reportedly positive control (Majin), all of which are exclusively expressed in the mouse testes by in silico screening. The male germline-specific expressions of the selected genes were confirmed by quantitative RT-PCR and existing single-cell RNA sequencing data. We directly generated bi-allelic knockout (KO) mice for each candidate by the triple-target CRISPR-based method. The reproductive phenotypes of the CRISPR founder males were examined by in vitro fertilization (IVF), computer assisted sperm motility analysis (CASA), and morphological/histological examinations. [Results] The embryonic or postnatal development of the founders was not influenced by the CRISPR injection. Spermatogenesis appeared to be completed in all the KO mice at the histological level. However, spermatozoa of Cox7b2 KO and Gm773 KO mice exhibited severe defects in fertilization by IVF. Cox7b2 KO spermatozoa showed extremely poor motility, while Gm773 KO spermatozoa showed normal motility. Morphologically, Cox7b2 KO spermatozoa showed a severe abnormality in their head and midpiece. Thus, our genetic screen identified Cox7b2 and Gm773 as critical factors for proper sperm differentiation and functions. Further analysis will reveal novel molecular mechanisms underlying spermatogenesis.