Lipid Nanoparticle-Mediated Delivery of CRISPR-Cas9 Components for Genome Editing.

Lipid nanoparticles (LNPs) are a clinically validated nonviral platform for the delivery of CRISPR-associated components. Composed of ionizable lipids, phospholipids, cholesterol, and PEG-lipids, LNPs enable the efficient encapsulation, protection, and cytosolic delivery of therapeutic cargo such as DNA, RNA, or proteins. The clinical relevance of LNPs has already been shown by multiple FDA-approved therapies, including siRNA-based treatments and mRNA vaccines. Compared with viral vectors, LNPs offer several advantages, including reduced immunogenicity, absence of genomic integration, scalable manufacturing, and flexibility in cargo size, while supporting transient expression, which is desirable for genome editing applications. However, challenges remain, including limited tissue specificity and inefficient endosomal escape. Recent advances in lipid chemistry optimization and surface modification have improved delivery performance. Among available formulation techniques, microfluidic mixing has emerged as a preferred method due to its reproducibility, scalability, and precise control over particle properties. This protocol describes a standardized microfluidic workflow for reproducible LNP formulation, providing practical guidance on lipid preparation, nanoparticle assembly, and quality control.