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

Most Sumona Akter,Shogo Matoba,Masashi Hada,Atsuo Ogura,Daiki Shikata,Gen Watanabe

doi:10.1038/s41598-021-94851-9

Abstract

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.

Highlights

Male germ cells undergo complex developmental processes eventually producing spermatozoa through spermatogenesis, the molecular mechanisms remain largely elusive
Twenty-nine somatic cell nuclear transfer-reprogramming resistant genes (SRRGs) were identified by the comparison of DNA methylome and transcriptome between fertilized blastocysts and somatic cell nuclear transfer (SCNT) blastocysts (DNA hypermethylated at the promoter [absolute methylation level > 5%] and transcriptionally repressed in SCNT blastocysts [Fold change > 2]; see Fig. 1a and Supplementary Table S1 online)
Faiml showed the highest expression in post-meiotic round or elongated spermatids. These results indicate that the six SRRGs, including Majin, are highly expressed in testicular germ cells, and suggest that SRRGs may play critical roles in mouse spermatogenesis

Summary

Introduction

Male germ cells undergo complex developmental processes eventually producing spermatozoa through spermatogenesis, the molecular mechanisms remain largely elusive. The groups of Ikawa and Matzuk have been performing extensive mouse genetic screening using the CRISPR/Cas[9] system and have identified many genes that are essential for male fertility or spermatogenesis[14,15,16,17] They and others found that the great majority of testis-expressed genes are individually dispensable for spermatogenesis or male fertility[14,18,19,20,21,22], which hampers the efficient identification of physiologically critical genes. We have previously performed a comprehensive comparison of transcriptome and epigenome between normally fertilized blastocysts and SCNT-generated blastocysts to identify SCNT-reprogramming resistant genes (SRRGs; Fig. 1a)[25] This list of SRRGs was highly enriched with the genes known to be essential for spermatogenesis in mouse KO models such as Asz[126], Tex1227, Slc25a3128, Tex10115,29, Mael[30], or Majin[31]. These included multiple X-linked genes with a high copy number of family genes (Mage and Xlr family) that are difficult to disrupt using CRISPR/Cas[9] simultaneously, some were unique genes, which indicates that the genetic screen by CRISPR/Cas[9] targeting is achievable

Methods

Results

Conclusion