Abstract: Light-inducible regulatory proteins are powerful tools to interrogate the fundamental mechanisms driving cellular behavior. To this end, genetically encoded photosensory domains fused to split proteins can tightly modulate protein activity and gene expression. While light-inducible split protein systems have performed well individually, few multichromatic and orthogonal gene regulation systems exist in mammalian cells. Existing multi-input circuits are hampered by their type of regulation and the scale of possible outputs given the number of wavelengths. I will address this limitation by creating a library of red and blue light-inducible split recombinases and the first suite of split Cas13 ribonucleases. Site specific recombinases provide permanent and transgene outputs, while Cas13 ribonucleases will provide a complementary approach to modulate reversible analog transcriptomic outputs. The multiplexed optogenetic tools developed in this proposal will be transformative for understanding the role of multiple interacting genes in endogenous signaling networks. I will leverage the ability of C2C12 myoblasts to differentiate into osteoblasts or myotubes and encode an illumination-dependent cell fate switch. While C2C12 cells have been a fundamental model in studying how differentiation is impacted by mechanical cues and growth factors, this work will explore spatiotemporal control of optogenetic regulatory proteins in order to direct cell fate.