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

Abstract

SummaryThe genetic basis of naive pluripotency maintenance and loss is a central question in embryonic stem cell biology. Here, we deploy CRISPR-knockout-based screens in mouse embryonic stem cells to interrogate this question through a genome-wide, non-biased approach using the Rex1GFP reporter as a phenotypic readout. This highly sensitive and efficient method identified genes in diverse biological processes and pathways. We uncovered a key role for negative regulators of mTORC1 in maintenance and exit from naive pluripotency and provided an integrated account of how mTORC1 activity influences naive pluripotency through Gsk3. Our study therefore reinforces Gsk3 as the central node and provides a comprehensive, data-rich resource that will improve our understanding of mechanisms regulating pluripotency and stimulate avenues for further mechanistic studies.

Zfp281 orchestrates interconversion of pluripotent states by engaging Ehmt1 and Zic2

“…We hypothesized that mechanisms common to differentiation and de‐differentiation may be encoded in genes that both promote exit from the naïve ESC state and impair reprogramming of EpiSCs. ESC differentiation drivers have been determined in several genetic loss‐of‐function screens (Guo et al , ; Westerman et al , ; Betschinger et al , ; Leeb et al , ; Li et al , ), but it is unknown whether those also inhibit reprogramming of EpiSCs into naïve pluripotency. We therefore set out to systematically identify reprogramming roadblocks using a large‐scale endoribonuclease‐prepared small interfering RNA (esiRNA) loss‐of‐function screen (Ding et al , ).…”

“…Note that esiRNAs targeting Mll1 (Zhang et al , ) and Mbd3 (Rais et al , ) were not included in our library and that Otx2 (Acampora et al , ) scored below the significance threshold. Screen hits with negative (blue) and positive (red) Z scores (red), and Tcf7l1 (green) are highlighted. Comparison of reprogramming screen hits with two ESC differentiation screens (Betschinger et al , ; Li et al , ). Empty and full circles indicate genes recovered in one and both ESC differentiation screens, respectively. Number of Epi‐iPSC colonies derived from 796.4 EpiSCs transfected with indicated siRNAs, stimulated with Gcsf and 2i for 4 days, and selected with puromycin.…”

“…Screen hits with negative (blue) and positive (red) Z scores (red), and Tcf7l1 (green) are highlighted. C Comparison of reprogramming screen hits with two ESC differentiation screens (Betschinger et al, 2013;Li et al, 2018). Empty and full circles indicate genes recovered in one and both ESC differentiation screens, respectively.…”

“…Screen replicates are presented in Fig EV1B. Average Z scores > 2 were considered as screen hits, identifying 146 genes ( Fig 1B). We quantified their role in exit from the ESC state by extracting primary data for these 146 genes from two previous ESC differentiation studies (Betschinger et al, 2013;Li et al, 2018), and computing Z scores on this subset (Table EV1). Sixty-seven and 129 of the 146 genes were mapped on results from Betschinger et al (2013) and Li et al (2018), respectively, resulting in mapping of 130 genes in total.…”

Ubiquitin Dynamics in Stem Cell Biology: Current Challenges and Perspectives

“…A number of genetic screens and analyses were conducted to identify and characterize DUBs and E3 Ub ligases essential for stem cell self‐renewal and cell fate choice . This has been facilitated by the production of haploid human embryonic stem cells and the development of sophisticated genetic screening assays based on the CRISPR/Cas9 technology .…”