Leydig cells (LCs) are distributed between the seminiferous tubules of the testis. They are the primary source of androgens, which are essential for the physical and mental well-being of men as well as the beginning of the puberty process [[1], [2], [3]]. Male hypogonadism can cause a series of clinical symptoms such as depressed mood, increased fatigue, weight loss and so on [2,4,5]. Although testosterone replacement therapy can play an effective role, long-term use of androgen can lead to heart disease, stroke, prostate cancer and other risks [6].

Transplantation of LCs has been shown to sustain long-term testosterone production and to preserve in vivo physiological patterns of the hormone [3,7,8]. Therefore, LCs transplantation has a promising potential to treat androgen deficiency. However, the absence of seed cells is the actual barrier impeding the implementation of LCs transplantation [1]. Stem cells (SCs) from a wide variety of origins have been induced to differentiate into LCs under controlled conditions in the past decade [[9], [10], [11]]. However, the SCs-inducing strategy is rather time-consuming and exists ethical issues [12,13]. Our previous studies have demonstrated that SCs derived from human umbilical cord tissues can differentiate into LCs by appropriate combination of various factors for several weeks, but it was very inefficient [14,15]. We have effectively turned HFFs into Leydig-like cells by a novel technique, the CRISPR/dCas9VP64 system, but the reprogramming efficiency was around 7%, and therefore further optimization is needed.

Herein, we describe an optimized CRISPR/dCas9 system that can enhance the ability to activate endogenous target genes by histone epigenetic modification [16]. The p300, a highly conserved acetyltransferase, controls many cellular processes [17,18]. The dCas9p300, an optimized CRISPR/dCas9 system, not only potentiated transcriptional activation by directly inducing epigenetic remodeling, but also facilitated generation of testosterone-producing Leydig-like cells. Here we observed that this dCas9p300 system displayed notably higher transactivation levels than the dCas9VP64 based on similar expression levels of dCas9 fusion proteins. Furthermore, the dCas9p300-mediated Leydig-like cells not only significantly expressed the steroidogenic biomarkers, but also produced more testosterone with or without LH treatment than the dCas9VP64-mediated. Similar to the features of targeted transcriptional activation caused by the dCas9p300, preferential enrichment in H3K27ac at the promoters was only identified with dCas9p300 treatment. Accordingly, the dCas9p300, optimized CRISPR/dCas9 system, may help to obtain sufficient Leydig-like cells closer to mature LCs for treating of androgen deficiency.