Myc and Dnmt1 impede the pluripotent to totipotent state transition in embryonic stem cell [CRISPR-Cas9 screen]

Totipotency is defined as the ability of a cell to generate all the cell types of an organism, including those of the extraembryonic tissues. Unlike pluripotency, the molecular features and the establishment of totipotency are poorly understood. In mouse embryonic stem cell (ESC) culture, a small percentage of cells transits into a totipotent state by expressing a group of genes that are only expressed in 2-cell-stage embryos. To understand how this transition takes place, we performed single cell RNA-seq analysis which revealed a two-step transcriptional reprogramming process characterized by downregulation of pluripotent genes in the first step and upregulation of the 2-cell embryo-specific genes in the second step. To identify factors controlling the transition process, we performed a CRISPR/Cas9-mediated genetic screen which revealed Myc and Dnmt1 as two factors preventing the transition. Mechanistic studies demonstrate that Myc prevents down-regulation of the genes in the first step, while Dnmt1 impedes gene activation in the second step. Collectively, our study reveals insights into the mechanism underlying establishment and regulation of totipotent state in ESCs. We performed a genome-wide CRISPR screen by transducing the ES cells containing LTR-tdTomato and inducible Dux transgene with a mouse CRISPR library expressing 79,632 gRNAs targeting 19,674 genes. After 24h of Dux induction, 2C+ and 2C- cell populations were purified for sequencing to determine the relative abundance of sgRNAs.