An inducible dual up/down gene regulation CRISPR system to study neuronal activity and regeneration - ABSTRACT The dynamic processes of axon regeneration and neuronal repair are governed by an intricate network of genetic pathways that involve multiple genes to be activated and repressed, but a full comprehension of how these genes interact remains elusive. While many individual genes that influence regeneration have been identi=ed, we do not yet have an approach for simultaneously and inducibly activating and repressing gene expression in neurons, especially in the context of dynamic process of neuronal regeneration during injury. An additional challenge is obtaining suf=cient temporal control over when each gene is activated or repression due to the dynamic nature of these processes. To address these unmet needs, we propose the development of a novel synthetic biology platform consisting of an inducible and multiplexed dual gene modulation system that is capable of simultaneous and sequential gene activation and suppression to characterize genetic interactions and therefore enhance neuronal regeneration following injury. Our system will take place in human induced pluripotent stem cell (iPSC) lines that can be effectively induced into neurons (iNs). By targeting speci=c groups of genes known to regulate axon regeneration for activation and repression at the same time, we will characterize how the system can be used to reveal multi-gene contributions in promoting neuron regeneration. Our approach leverages recent advances in CRISPR-based multiplexed tools, namely hyperdCas12a and Cas13d, both capable of processing own highly compact guide RNA arrays to target multiple loci with high precision, while Cas12a targets DNA for activation and Cas13d targets mRNA for repression. We will engineer a system containing both hyperdCas12a and Cas13 capable of inducible and multiplexed simultaneous gene activation and repression with temporal control. In Aim 1, we will develop an inducible and multiplexed system in neurons that are differentiated from iPSCs and characterize our system’s effectiveness in enhancing neuron regeneration beyond single gene perturbations. In Aim 2, we will utilize multiplexing technology to de=ne an epistatic network of interacting genes (50 genes) across different stress conditions in neurons. Subsequent neuron injury-based functional validations of these computation results will determine the roles of these interactions in neural protection and regeneration. Our proposed project will establish a novel and impactful platform for inducible and dual multiplexed gene regulation in neurons. Our system offers an effective technology to target multiple genes dictating neural regeneration potential, thus providing a means of achieving axon regeneration beyond what has been reported. Our work addresses unmet needs in synthetic neuroscience and provide a unique strategy for studying neuron regeneration. Ultimately, our work will help the broader scienti=c community by 1) providing a broadly useful tool to activate and repress multiple genes in an ‘all-in-one’ system; and 2) delineate gene interactions and pathways in neurons to influence neural regeneration.
