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Basic Science and Pathogenesis.

Daniel M Ramos1, Matthew P Nelson1, Liz Calzada1

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Summary
This summary is machine-generated.

We developed an inducible CRISPRi system for gene knockdown in neurons derived from induced pluripotent stem cells (iPSCs). This tool enables the discovery of neuron-specific disease modifiers and potential therapeutic targets.

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Area of Science:

  • Stem cell biology
  • Genome engineering
  • Neuroscience

Background:

  • The iPSC Neurodegenerative Disease Initiative (iNDI) is a large project modeling over 100 Alzheimer's disease-related dementia (ADRD) mutations using induced pluripotent stem cells (iPSCs).
  • CRISPR interference (CRISPRi) screens are valuable for identifying disease mechanisms, but current tools are not optimized for iPSC-derived neurons (iNeurons).

Purpose of the Study:

  • To develop a Cre-lox inducible CRISPRi system (CRISPRi-Cre) for targeted gene knockdown in postmitotic iNeurons.
  • To enable the identification of neuron-specific modifiers relevant to neurodegenerative diseases.

Main Methods:

  • A Cre-inducible CRISPRi system was constructed by modifying a dCas9 transcriptional repressor plasmid with a floxed STOP cassette.
  • The system's functionality was validated in iPSCs and iNeurons using flow cytometry and sgRNAs.
  • Genome-wide CRISPRi survival screens were performed in iNeurons to assess the system's broad applicability.

Main Results:

  • The CRISPRi-Cre system demonstrated inactive dCas9 without Cre, and potent gene knockdown upon Cre delivery to iNeurons.
  • Genome-wide screens using CRISPRi-Cre identified known and novel neuron-specific gene regulators.
  • The system successfully uncovered neuron-specific hits not found in previous CRISPRi screens.

Conclusions:

  • A robust Cre-inducible CRISPRi system was developed for post-mitotic gene knockdown in iPSC-derived neurons.
  • This tool facilitates the discovery of neuron-specific disease mechanisms, modifiers, and therapeutic targets.
  • The CRISPRi-Cre system enhances the study of neurodegenerative diseases in relevant cellular models.