Cell-Penetrating Peptide Delivery for CRISPR Applications.

Abstract

Cell-penetrating peptides (CPPs) provide a modular nonviral strategy for intracellular delivery of CRISPR/Cas9 components, particularly preassembled Cas9 ribonucleoprotein (RNP) complexes. This chapter summarizes key principles of CPP-mediated Cas9 RNP delivery, including noncovalent complexation and covalent CPP-Cas9 conjugation strategies. A representative protocol is provided for the preparation and evaluation of Cas9-K5 RNP, including in vitro uptake and reporter-editing assays, nucleofection-based functional controls, local intracranial delivery, immunofluorescence staining, and quantitative analysis of reporter activation in brain tissue.