3D GelMA-Printing of Pancreatic Endocrine Constructs via Magnetic Chitosan Nanoparticle-Mediated CRISPR/dCas9a Delivery: A Proof-of-Concept Study

Abstract

Diabetes mellitus affects nearly half a billion people worldwide and is primarily caused by the loss of insulin-producing cells, which are essential for maintaining normal blood glucose levels. In this study, we optimized a CRISPR activation (CRISPRa) system based on deactivated Cas9 (dCas9) to transfect HEK293 cells, which were subsequently differentiated into functional α-, β-, and δ-like cells as an engineering proof-of-concept. The CRISPRa system targeted multiple genes, including NGN3, NKX6.1 + MAFA, and PDX1, successfully inducing cell differentiation as confirmed by immunostaining. These cells were encapsulated in a 3D-printed GelMA disk, and the secretion of glucagon, insulin, and somatostatin was monitored for 30 days.