Abstract

Abstract Dendritic cells (DCs) are critical regulators of T cell immunity, with immense therapeutic potential against tumors and autoimmune diseases. Efficient gene editing in DCs is crucial for understanding their regulatory mechanisms and maximizing their therapeutic efficacy. However, DCs are notoriously difficult to transfect, posing a major bottleneck for conventional DNA and RNA‐based editing approaches. Microneedle‐mediated injection of Cas9/sgRNA ribonucleoprotein (RNP) directly into the nucleus, akin to gene editing in reproductive cells, offers promise but suffers from limitations in scalability. Here, an intranuclear delivery system using a hollow nanoneedle array (HNA) combined with nano‐electroporation is developed. The 2 µm‐high HNA physically reaches the nucleus, positioning the nuclear envelope and plasma membrane in close proximity at the tip. Transient electronic pulses then induce simultaneous perforations across all 3 membranes, enabling direct RNP delivery into the nucleus. This HNA‐based system achieves efficient knockout of genes like PD‐L1 in primary DCs, demonstrating its potential as a powerful tool for gene editing in DCs and other hard‐to‐transfect cells.