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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.