A modular system for programming multistep activation of endogenous genes in stem cells

Although genomes encode instructions for mammalian cell differentiation with rich syntactic relationships, existing methods for genetically programming cells have modest capabilities for stepwise regulation of genes. Here, we developed a sequential genetic system that enables transcriptional activation of endogenous genes in a preprogrammed, stepwise manner. The system relies on the removal of an RNA polymerase III termination signal to induce both the transcriptional activation and the DNA endonuclease activities of a Cas9-VPR protein to effect stepwise progression through cascades of gene activation events. The efficiency of the cascading system enables a new dimension for cellular programming by allowing the manipulation of the sequential order of gene activation for directing the differentiation of human stem cells.