Development of a human iPSC tool kit containing the CRISPR/CAS based Castilio system to test complex genetic circuits in cell differentiation

Pluripotent cells possss the ability to self-renew or differentiate into somatic cell types based on changes in gene-expression. Human induced pluripotent stem cells (hiPSCs) are somatic cells reprogrammed to become pluripotent, and can be used in vitro to help uncover differentiation processes for potential in vivo therapeutic uses. However, genes primarily responsible for differentiation are still largely unknown. Gene-editing tools that can moduylate gene-expression of multiple genes simultaneously are requred to better understand gene networks that drive differentiation. The CRISPR/Cas based Casilio tool kit comprises an enzymatically dead Cas9 (dCas9), a RNA-binding Pumilio/FBF (PUF) protein(s) motif fused to transcription modulators, and a reprogrammable guide RNA appended with a PUF binding site (gRNA-PBS) that can direct, and localize, the dCas9 and Pumilio complexes to desired DNA sequences in the genome. The modular nature of the Casilio system provides advantages over the traditional CRISPR transcriptional inhibitory or activating methods by enabling the concomitant repression of certain genes while activating others. In this proof-of-concept experiment, we genetically engineer a novel hiPSC line to endogenously express Casilio proteins through lentiviral-based infection, and determine if the overexpression of the endogenous transcription factor NEUROGENIN-2 can drive hiPSCs from an immortal pluripotent state to a differentiated neuron in vitro. Our goal is for this novel transgenic hiPSC line, expressing all Casilio components excluding the reprogrammable gRNA-PBS, to enable researchers to quickly test complex gene-expression networks and their functions in cellular processes, tapping into the potential for in vivo iPSC-derived cell therapeutic uses