Focused Ultrasound and Microbubble-Mediated Delivery of CRISPR-Cas9 Ribonucleoprotein to Human Induced Pluripotent Stem Cells

Kyle Hazel; Davindra Singh; Stephanie He; Zakary Guertin; Mathieu C Husser; Brandon Helfield

doi:10.1016/j.ymthe.2025.01.013

Focused Ultrasound and Microbubble-Mediated Delivery of CRISPR-Cas9 Ribonucleoprotein to Human Induced Pluripotent Stem Cells

Mol Ther. 2025 Jan 9:S1525-0016(25)00017-6. doi: 10.1016/j.ymthe.2025.01.013. Online ahead of print.

Authors

Kyle Hazel¹, Davindra Singh¹, Stephanie He¹, Zakary Guertin¹, Mathieu C Husser¹, Brandon Helfield²

Affiliations

¹ Department of Biology, Concordia University, 7141 Sherbrooke St. W H4B 1R6, Montreal, Canada.
² Department of Biology, Concordia University, 7141 Sherbrooke St. W H4B 1R6, Montreal, Canada; Department of Physics, Concordia University, 7141 Sherbrooke St. W H4B 1R6, Montreal, Canada. Electronic address: brandon.hefield@concordia.ca.

PMID: 39797397
DOI: 10.1016/j.ymthe.2025.01.013

Abstract

CRISPR-Cas9 ribonucleoproteins (RNPs) have been heavily considered for gene therapy due to their high on-target efficiency, rapid activity and lack of insertional mutagenesis relative to other CRISPR-Cas9 delivery formats. Genetic diseases such as hypertrophic cardiomyopathy currently lack effective treatment strategies and are prime targets for CRISPR-Cas9 gene editing technology. However, current in-vivo delivery strategies for Cas9 pose risks of unwanted immunogenic responses. This proof-of-concept study aimed to demonstrate that focused ultrasound (FUS) in combination with microbubbles can be used to deliver Cas9-sgRNA (single guide RNA) RNPs and functionally edit human induced pluripotent stem cells (hiPSCs) in-vitro, a model system that can be expanded to cardiovascular research via hiPSC-derived cardiomyocytes. Here, we first determine acoustic conditions suitable for the viable delivery of large proteins to hiPSC with clinical Definity® microbubble agents using our customized experimental platform. From here, we delivered Cas9-sgRNA RNP complexes targeting the EGFP (enhanced green fluorescent protein) gene to EGFP-expressing hiPSCs for EGFP knockout. Simultaneous acoustic cavitation detection during treatment confirmed a strong correlation between microbubble disruption and viable FUS-mediated protein delivery in hiPSCs. This study shows, for the first time, the potential for an FUS-mediated technique for targeted and precise CRISPR-Cas9 gene editing in human stem cells.