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

Introduction

Mammalian male germ cells undergo unique and complex differentiation processes involving sex determination, epigenetic reprogramming, cell migration, and spermatogenesis finally generating motile spermatozoa^{1, 2–3}. Spermatozoa, or haploid spermatids, are solely responsible for the transmission of male genetic information to the next generation via fertilization with oocytes. In humans, it has been estimated that infertility affects 8–12% of couples globally and male factors play a primary or contributing cause in 50% of these couples⁴. Although a variety of factors could cause infertility in men, the primary cause resides in germ cell development or differentiation processes that lead to the absence of spermatozoa or abnormal spermatozoa^4,5. Although assisted reproductive techniques such as intracytoplasmic sperm injection or round spermatid injection could partly overcome these problems^6,7, it is important to understand the mechanisms of infertility to develop new technologies to support reproduction. Despite such importance, the mechanisms of these dynamic processes of male germ cell development remain elusive.

A previous in silico survey of gene expression datasets estimated that more than 2,300 genes were predominantly expressed in male germ cells⁸. Indeed, dozens of genes have been identified that play essential roles in male germ cell development or fertility mainly using mouse genetic models. Recently, the genome-editing technology, CRISPR/Cas9^{9, 10–11}, has enhanced the speed of genetic screening^12,13. For example, the groups of Ikawa and Matzuk have been performing extensive mouse genetic screening using the CRISPR/Cas9 system and have identified many genes that are essential for male fertility or spermatogenesis^{14, 15, 16–17}. However, they and others also 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. Therefore, it is ideal to have a unique strategy that efficiently narrows down functionally important candidates before performing the real genetic screen.

Germ cells separate from somatic cell lineages at the early stage of embryonic development²³. Thus, when the somatic cell genome is transferred to enucleated oocytes by somatic cell nuclear transfer (SCNT), the reconstructed genome skips the epigenetic reprogramming steps that normally occur during germ cell development²⁴. We have previously performed a...