Hours of sources.
One expert brief.

Core takeaways

• Delivery is the bottleneck, not the nuclease. Editing efficiency in vitro routinely exceeds 80%. In vivo outcomes collapse to single digits outside liver and eye. The limiting variable is cargo arriving intact in the right cell type, not Cas9 activity.
• Three vector families, three failure modes. AAV fails on cargo size and pre-existing immunity. Lipid nanoparticles fail on extrahepatic targeting. Virus-like particles and electroporation fail on scale and manufacturing consistency.
• Transient beats persistent for safety. Ribonucleoprotein and mRNA payloads clear within days, which shortens off-target exposure. AAV-delivered Cas9 can persist for months, and persistence is now treated as a liability rather than a feature.

Key concepts

Tropism

The tissue preference of a vector, set mostly by capsid surface residues in AAV and by ionizable lipid chemistry plus protein corona in LNPs.

Packaging limit

AAV carries ~4.7 kb of single-stranded DNA. SpCas9 plus promoter and guide occupies nearly all of it, which forces dual-vector splits or smaller nucleases.

Protein corona

The layer of serum proteins that adsorbs to a nanoparticle in circulation. ApoE adsorption routes LNPs to hepatocytes via LDL receptor, which is why liver is the default destination.

RNP delivery

Pre-assembled Cas9 protein plus guide RNA. No transcription or translation step, activity peaks within hours, clearance within roughly 72 hours.

Facts worth memorising

• AAV packaging capacity: ~4.7 kb single-stranded. SaCas9 (3.2 kb) fits with a guide; SpCas9 (4.1 kb) does not fit with regulatory elements.
• LNP biodistribution without targeting ligands: >90% of dose to liver within 30 minutes of IV administration.
• Neutralizing antibody prevalence against AAV2 in adults: roughly 30–60%; AAV5 is lower, which is why AAV5 appears in systemic programmes.
• Approved editing therapies to date use ex vivo electroporation of RNP, sidestepping in vivo delivery entirely. Trial readout Q3

Process: selecting a vector

• Define the target cell type first. Vector selection is downstream of that decision, never upstream.
• Check cargo size against packaging limit. Over ~4.5 kb, AAV requires a dual-vector split or a compact nuclease.
• Screen for pre-existing neutralizing antibodies if the route is systemic AAV. Titre above threshold excludes the patient.
• Select payload format: RNP for shortest exposure, mRNA for moderate, DNA for sustained expression where persistence is acceptable.