Parallel genome-scale CRISPR-Cas9 screens uncouple human pluripotent stem cell identity versus fitness

Pluripotent stem cells have remarkable self-renewal capacity: the ability to proliferate indefinitely while maintaining the pluripotent identity essential for their ability to differentiate into almost any cell type in the body. To investigate the interplay between these two aspects of self-renewal, we perform four parallel genome-scale CRISPR-Cas9 loss-of-function screens interrogating stem cell fitness in hPSCs and the dissolution of primed pluripotent identity during early differentiation. These screens distinguish genes with distinct roles in pluripotency regulation, including mitochondrial and metabolism regulators crucial for stem cell fitness, and chromatin regulators that control pluripotent identity during early differentiation. We further identify a core set of genes controlling both stem cell fitness and pluripotent identity, including a network of chromatin factors. Here, unbiased screening and comparative analyses disentangle two interconnected aspects of pluripotency, provide a valuable resource for exploring pluripotent stem cell identity versus cell fitness, and offer a framework for categorizing gene function.