Nanopore Electroporation: A New Delivery Method Within the Field of Epigenetic Editing

ABSTRACT Epigenetic modifications influence gene expression and contribute to type 2 diabetes (T2D), but establishing causality requires targeted modulation of specific genes. CRISPR‐dCas9‐based tools offer this potential, yet β‐cells are notoriously difficult to transfect, and efficient, non‐viral delivery methods are lacking. Here, we developed nanopore‐mediated electroporation to deliver a CRISPR interference (CRISPRi) system to clonal INS1 β‐cells, achieving targeted downregulation of insulin expression. Cells were seeded atop a nanopore substrate with CRISPRi plasmids in solution below. Mild electric pulses generated transient nanoscale pores in the membrane, enabling electrophoretic delivery of plasmids into the cytosol while preserving high cell viability. The CRISPRi system comprised the transcriptional repressor Krueppel‐associated Box Domain (KRAB) fused to an inactive Cas9 (dCas9), guided to the transcription start site of the insulin‐1 gene ( Ins1 ) by a single guide RNA (sgRNA). After transfection, Ins1 expression was significantly reduced, demonstrating effective modulation of gene expression in this difficult‐to‐transfect cell type. This nanopore electroporation approach provides a robust, safe, and efficient platform for delivering CRISPR‐dCas9‐based epigenetic editors in pancreatic β‐cells. By enabling precise gene regulation, it opens avenues for mechanistic studies of epigenetic contributions to T2D and potentially other challenging cell systems.