Cas9 Delivery: Focused Ultrasound and Nanotechnology Approaches

CRISPR-Cas9 enables precise genome editing but faces delivery challenges, including poor cellular uptake, off-target effects, and immune activation. Focused ultrasound (FUS) and nanotechnology-based carriers offer synergistic, non-invasive solutions, enhancing delivery efficiency, tissue penetration, and targeting.

A systematic search (January 2010-March 2025) of PubMed, Web of Science, Scopus, Embase, and Google Scholar databases, preprint servers, and clinical trial registries identified studies employing FUS, nanoparticles (NPs), or combined approaches for CRISPR-Cas9 delivery. Eligible studies reported outcomes on efficiency, specificity, safety, or therapeutic effect in cell lines, animal models, or humans. Data extraction and quality assessment were performed by two independent reviewers. Narrative synthesis and meta-analysis were conducted where appropriate.

Forty-eight studies (21 in vitro, 19 in vivo, 8 clinical) met the inclusion criteria. NPmediated delivery achieved ~75% editing efficiency and enhanced stability versus viral or naked methods. FUS increased tissue penetration by threefold and reduced systemic toxicity. Combined FUS-NP systems improved blood-brain barrier penetration and maintained low off-target rates (>80% of studies) with minimal immune responses.

Hybrid FUS-NP delivery platforms address key barriers in CRISPR-Cas9 therapy, particularly for neurological, oncological, and genetic disorders. Nonetheless, heterogeneity in NP composition, ultrasound settings, and target genes limits direct comparability, and long-term clinical safety remains underexplored.

FUS-assisted nanotechnology provides a promising non-viral strategy for precise, safe, and targeted CRISPR-Cas9 delivery. Standardization of technical parameters and regulatory frameworks are essential for successful clinical translation.