Genome-wide CRISPR-KO Screen Uncovers mTORC1-Mediated Gsk3 Regulation in Naive Pluripotency Maintenance and Dissolution

摘要

class="head no_bottom_margin" id="sec1title">IntroductionMouse embryonic stem cells (mESCs) are derived from the inner cell mass of blastocyst-stage embryos and can be indefinitely propagated while maintaining the ability to differentiate into all three germ layers. They have served not only as a platform for genome manipulation and production of transgenic mice but also as an essential model system to study the molecular mechanisms of self-renewal and differentiation. In particular, mechanisms that underpin the maintenance of pluripotency have been the subject of intense research, establishing the framework through which the pluripotent state is regulated by intrinsic and extrinsic factors. Intrinsically, the core transcription factors Pou5f1, Sox2, and Nanog act together with accessory factors Esrrb, Klf2, and Tfcp2l1 to consolidate the pluripotent identity (). Extrinsically, leukemia inhibitory factor (LIF)-STAT3 signaling plays a key role to sustain pluripotency () and Wnt signaling cooperates to suppress differentiation (), whereas FGF-MAPK signaling is essential for mESCs to initiate differentiation (). In uncovering these basic principles, culture conditions that permit the preservation of pluripotency via dual inhibition of MEK and Gsk3 kinases (termed 2i) were established (). mESCs cultured with 2i closely resemble epiblasts in pre-implantation embryos, sharing transcriptomic and epigenomic features that reflect the ground or naive state of pluripotency (, ).As the mechanisms of pluripotency maintenance have become clearer, research focus has shifted toward understanding how the exit from pluripotency and initiation of lineage specification are achieved. In response to differentiation cues, mESCs must resolve the naive pluripotency network and initiate transcriptional events that drive the progression through an intermediate or formative state to the primed state (). One key factor regulating this process is Tcf7l1, which is a transcriptional suppressor and colocalizes with Pou5l1 and Sox2, thereby counteracting their transcriptional activation and suppressing the intrinsic pluripotency program (). Conversely, loss of Tcf7l1 resulted in upregulation of Nanog, severely delaying the onset of differentiation (). The activity of Tcf7l1 is subject to regulation by Wnt signaling and thus depends on Gsk3 activity. Inhibition of Gsk3 results in the nuclear translocation of β-catenin, which upon binding to Tcf7l1, abrogates its suppressor activity (, ). This is a clear example of how extrinsic signaling dictates the dissolution of the core pluripotency network. Although reverse and forward genetic approaches have been successful in identifying such factors (, , , href="#bib29" rid="bib29" class=" bibr popnode">Leeb et al., 2014, href="#bib44" rid="bib44" class=" bibr popnode">Pereira et al., 2006), the full repertoire of genes and pathways involved in this process remains elusive.The CRISPR-Cas system is the defense machinery found in a range of bacterial and archaea species (href="#bib33" rid="bib33" class=" bibr popnode">Makarova et al., 2015). Among them, the CRISPR-Cas9 system derived from Streptococcus pyogenes is most extensively characterized (href="#bib21" rid="bib21" class=" bibr popnode">Jinek et al., 2012, href="#bib22" rid="bib22" class=" bibr popnode">Jinek et al., 2014, href="#bib38" rid="bib38" class=" bibr popnode">Nishimasu et al., 2014, href="#bib52" rid="bib52" class=" bibr popnode">Sternberg et al., 2014) and has been adapted into versatile genetic tools (href="#bib1" rid="bib1" class=" bibr popnode">Adli, 2018). The key advantage of the CRISPR-Cas9 system is the high consistency and efficiency in generating targeted gene knockouts, which has enabled us and others to carry out genome-scale CRISPR-knockout (KO) screening in mammalian cells (href="#bib27" rid="bib27" class=" bibr popnode">Koike-Yusa et al., 2014, href="#bib48" rid="bib48" class=" bibr popnode">Shalem et al., 2014, href="#bib57" rid="bib57" class=" bibr popnode">Wang et al., 2014). CRISPR-KO screening has shown superior detection sensitivity compared to RNAi screens (href="#bib12" rid="bib12" class=" bibr popnode">Evers et al., 2016), and its resolving power is evident in the unraveling of genetic dependencies in cancer cells (href="#bib18" rid="bib18" class=" bibr popnode">Hart et al., 2015, href="#bib55" rid="bib55" class=" bibr popnode">Tzelepis et al., 2016, href="#bib58" rid="bib58" class=" bibr popnode">Wang et al., 2017).Here we performed CRISPR-KO phenotypic screens to gain more in-depth insight and comprehensive understanding of the maintenance of and exit from naive pluripotency. The unbiased nature of CRISPR-KO screening revealed multiple genes and protein complexes whose functions have not previously been associated with pluripotency maintenance and/or differentiation. In particular, our screen revealed that regulation of Gsk3 activity is a key requirement in initiating differentiation. In addition, regulation of Gsk3 is mediated by Akt/mTOR signaling, subsequently linking nutrient and energy metabolism pathways to the exit from naive pluripotency. Our study therefore represents the most comprehensive account of the factors involved in the regulation of naive pluripotency, providing a key resource for further experimental interrogation.