Basic Science and Pathogenesis

The iPSC Neurodegenerative Disease Initiative (iNDI) is the largest-ever induced pluripotent stem cell (iPSC) genome engineering project, modeling over 100 ADRD mutations in high-quality isogenic human iPSCs. iNDI leverages unbiased CRISPRi screens as a powerful tool to identify fundamental mechanisms and modifiers of disease. However, current CRISPRi molecular tools are poorly optimized for use in iPSC-derived neurons (iNeurons). Here we develop a Cre-lox inducible CRISPRi system (CRISPRi-Cre), enabling gene knockdown upon Cre delivery to postmitotic iNeurons, and identification of neuron-specific, disease-relevant modifiers.

We modified a plasmid carrying a potent Zim3-dCas9 transcriptional repressor to include a strong floxed STOP cassette upstream of the Zim3 start codon. We leveraged HaloTag-TDP43 and HaloTag-FUS iSPCs from the iNDI project paired with flow cytometry to validate leakiness and responsiveness to Cre in iPSCs and iNeurons treated with sgRNAs. We then performed a genome-wide CRISPRi survival screen in iNeurons to demonstrate broad functionality of this inducible CRISPRi system with over 20,000 sgRNAs. Finally, we use CRISPRi-Cre to identify neuron-specific regulators of neuronal activity in iNeurons.

We demonstrate that in the absence of Cre, dCas9 is inactive. Delivery of lentivirus-Cre to iNeurons activates dCas9, resulting in potent gene knockdown. In genome-wide CRISPRi screens, we show that CRISPRi-Cre identifies many of the same hits observed in screens using constitutive-active dCas9, and importantly uncovers novel neuron-specific hits not identified in previous CRISPRi screens.

Here, we developed a robust Cre-inducible CRISPRi system that enables post-mitotic gene knockdown in iPSC-derived neurons. Our CRISPRi screens identify neuron-specific hits, demonstrating the utility of our tool to help uncover disease-relevant mechanisms, modifiers, and potential therapeutic targets in relevant cell types.