Integrative multiomic analysis reveals how non-viral delivery system selection shapes CRISPR gene editing outcomes in stem cells.

Non-viral delivery avoids the immunogenicity and genomic integration risks of viral vectors but faces fundamental trade-offs between editing efficiency and cytotoxicity. Here, we present a comprehensive multiomic analysis of four non-viral CRISPR delivery modalities including cell-penetrating peptide- (CPP), lipid-, and polymer-based nanoparticles and electroporation; across mRNA and ribonucleoprotein (RNP) molecular formats.

We systematically evaluate each modality, demonstrating that lipid-based delivery achieved the highest editing rates at the cost of genomic instability, interferon pathway activation, and a pro-inflammatory shift in the MSC paracrine activity. Alternatively, CPPs yield moderate editing rates while reducing these unintended side-effects, whereas polymers and electroporation consistently yielded the lowest efficiencies. CRISPR molecular format and delivery method interacted in a stress-dependent manner, with RNP delivery reducing editing rates under high-stress systems while improving them in lower-stress modalities such as CPP and electroporation.