Keisuke Yamada
Engineering Cell-Penetrating Cas9 for Genome Editing in Primary Cells
Abstract
The CRISPR-Cas system has revolutionized genome editing and holds great therapeutic potential, but achieving both efficient and safe delivery into primary human cells remains a challenge. We previously developed Peptide-Assisted Genome Editing (PAGE), a strategy that enables direct Cas protein delivery with minimal toxicity. However, delivery of Cas9 ribonucleoproteins (RNPs) remains inefficient due to the strong negative charge of the Cas9-RNA complex. This project aims to improve RNP delivery through two strategies: (1) developing a high-throughput method to evaluate thousands of cell-penetrating peptide (CPP)-Cas9 RNPs, generating data to support predictive CPP design; and (2) engineering supercharged Cas9 variants with enhanced cell-penetrating ability, guided by computational modeling and experimental validation. By integrating protein engineering, deep learning, and scalable screening, this work will expand PAGE to a broader range of CRISPR tools in hard-to-transfect human cells.
Deeply grateful to receive the PhRMA Fellowship supporting my PhD studies. This honor boosts my confidence as a scientist and motivates me to contribute to advancing the field, give back to those who helped me grow and pay it forward.
