Parallel genome-wide CRISPR screens to identify state-dependent self-renewal regulators of mouse embryonic stem cells.

The pluripotency of embryonic stem cells (ESCs) is more accurately viewed as a continuous developmental process rather than a fixed state. However, the factors that play general or state-specific roles in regulating self-renewal in different pluripotency states remain poorly defined. In this study, parallel genome-wide CRISPR/Cas9 knockout (KO) screens were applied in ESCs cultured in the serum plus LIF (SL) and in the 2i plus LIF (2iL) conditions. The candidate genes were classified into seven groups based on their positive or negative effects on self-renewal, and whether this effect was general or state-specific for ESCs under SL and 2iL culture conditions. We characterized the expression and function of genes in these seven groups. The loss of function of novel pluripotent candidate genes Usp28, Zfp598, and Zfp296 was further evaluated in mouse ESCs. Consistent with our screen, the knockout of Usp28 promotes the proliferation of SL-ESCs and 2iL-ESCs, whereas Zfp598 is indispensable for the self-renewal of ESCs under both culture conditions. The cell phenotypes of Zfp296 KO ESCs under SL and 2iL culture conditions were different. Our work provided a valuable resource for dissecting the molecular regulation of ESC self-renewal in different pluripotency states.