Viral vectors may integrate into host cells and cause problems such as mutations, carcinogenesis, and immune responses.11 However, recombinant AAVs are non-replicating and tend to remain episomal without integrating into the genome, except in rare cases. These advantages allow their use for packaging CRISPR reagents into the AAV genome for cell editing.12 AAVs can also provide tissue-specific tropism due to the cell-specific serotypes of AAVs, have reduced immunogenicity compared to other viral vectors, and enable transient gene expression.13 However, challenges remain regarding efficiency and safety.
Non-viral delivery methods include the use of nanoparticles or liposomes to encapsulate ribonucleoprotein complexes.14–16 Nanoparticles made from engineered lipids or polymers can improve cell uptake, increase editing potency beyond what is achievable by protein alone, expand delivery beyond the liver, and potentially reduce immunogenicity compared to viral approaches.17 Another non-viral method uses messenger RNA (similar to RNA vaccines) to encode the CRISPR components, achieving transient and controlled expression without needing to deliver DNA.18
Lastly, the CRISPR system can be used without delivering the CRSIPR components directly into the body. Ex vivo therapy involves removing cells from the patient, editing them outside the body, and then returning them to treat genetic disorders like sickle cell anemia or enhance immune responses for cancers.
