UCSF

The directed differentiation of stem cells has a wide range of applications in fields such as regenerative medicine, in vitro cell biology, and disease modeling. Here, I present a CRISPR-based strategy to modulate target gene expression and direct cell fate. My system employs orthogonal nuclease-deactivated dCas9s derived from distinct bacterial species that when fused to transcriptional activator or repressor domains, enable the simultaneous activation and repression of specific target genes. By stably integrating these activating and repressing dCas9s into mouse embryonic stem cells (ESCs) to regulate the expression of specific transcription factors, I demonstrate an optimized strategy for the production of neurons. I show that this system increases the efficiency of neuronal induction relative to activation alone when genes that promote non-neuronal fates are simultaneously repressed. Additionally, using a different set of sgRNAs, I show that this system can be used to direct ESCs to a muscle cell fate. Altogether, my results establish a unique platform to program cell fate through the introduction of sgRNA cocktails to drive activation and repression of target genes.