Non-viral in vivo cytidine base editing in hepatocytes using focused ultrasound targeted microbubbles

CRISPR-Cas9-based genome editing technologies, such as base editing, have the potential for clinical translation, but delivering nucleic acids into target cells in vivo is a major obstacle. Viral vectors are widely used but come with safety concerns, while current non-viral methods are limited by low transfection efficiency. Here we describe a new method to deliver CRISPRCas9 base editing vectors to the mouse liver using focused ultrasound targeted microbubble destruction (FUTMD). We demonstrate, using the example of cytosine base editing of the Pde3b gene, that FUTMD-mediated delivery of cytosine base editing vectors can introduce stop codons (up to X2.5% on-target editing) in mouse liver cells in vivo. However, base editing specificity is less than one might hope with these DNA constructs. Our findings suggest that FUTMD-based gene editing tools can be rapidly and transiently deployed to specific organs and sites, providing a powerful platform for the development of non-viral genome editing therapies. Nonviral delivery also reveals greater off-target base exchange in vivo than in vitro.