Recombinant Fusion of Polyglutamic Acid Enhances Cell-Penetrating Peptide-Mediated CRISPR-Cas9 Delivery

Genome editing has been revolutionized by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein 9 (Cas9) technology. However, efficient intracellular delivery of Cas9 ribonucleoprotein (RNP) remains a major challenge, particularly in cells that are refractory to conventional transfection methods. Integrating viral vectors carry a risk of insertional mutagenesis, whereas physical methods such as electroporation are not well suited for in vivo or clinically relevant applications. Cell-penetrating peptides (CPPs) offer a promising alternative for direct RNP delivery, although their efficiency is often limited by endosomal entrapment and limited cytosolic release. In this study, we developed two recombinant CPP-fused Cas9 proteins, polyglutamate (PE)-trans-activator of transcription (TAT)-nuclear localization signal (NLS)-Cas9 (PETATCas9) and TAT-NLS-Cas9 (TATCas9), designed to improve solubility, membrane penetration, RNP complex formation, and nuclear targeting. PE tag in PETATCas9 was introduced to optimize charge balance and reduce nonspecific electrostatic interactions between TAT and guide RNA (gRNA), thereby enhancing RNP stability and delivery performance. Both recombinant proteins were expressed, purified, and assembled with gRNA into functional RNP complexes for delivery into SH-SY5Y neuroblastoma cells. The optimized CPP-Cas9 RNP system enabled efficient intracellular delivery and glucosylceramidase beta 1 (GBA1) gene editing in SH-SY5Y cells. These findings establish PETATCas9 as a scalable, transient, nonviral, and self-delivering RNP platform for CRISPR-based genome editing in cells that are refractory to conventional transfection methods.