ABSTRACT
CRISPR/Cas9 ribonucleoprotein (RNP) complexes with transient therapeutic activity and minimum off-target effects have attracted tremendous attention in recent years for genome editing and have been successfully employed in diverse targets. One ongoing challenge is how to transport structurally and functionally intact Cas9 protein and guide RNA molecules into cells efficiently and safely. Here we report a combinatorial library of disulfide bond-containing cationic lipidoid nanoparticles (LNPs) as carrier systems for intracellular Cas9/sgRNA delivery and subsequent genome editing. Nanoparticles with high efficacies of targeted gene knockout as well as relatively low cytotoxicities have been identified through in vitro screening. The in vivo biodistribution profiles were studied utilizing fluorescent dye labeled and RNP complexed LNPs. Results from this study may shed some light on the design of effective cationic lipidoids for intracellular delivery of genome editing platforms, as well as optimizing the nanoparticle formulations for further disease modeling and therapeutic applications.
Subject(s)
Biomimetic Materials/chemistry , CRISPR-Cas Systems/genetics , Drug Carriers/chemistry , Intracellular Space/metabolism , Nanoparticles/chemistry , Ribonucleoproteins/chemistry , Ribonucleoproteins/metabolism , Animals , Drug Carriers/pharmacokinetics , HeLa Cells , Hemolysis/drug effects , Humans , Lipids/chemistry , Mice , Ribonucleoproteins/genetics , Tissue DistributionABSTRACT
Protein based therapeutics with high specificities and low off-target effects are used for transient and accurate manipulation of cell functions. However, developing safe and efficient carriers for intracellular delivery of active therapeutic proteins is a long-standing challenge. Here we report a combinatorial library of chalcogen (O, S, Se) containing lipidoid nanoparticles (LNPs) as efficient nanocarriers for intracellular delivery of negatively supercharged Cre recombinase ((-30)GFP-Cre) and anionic Cas9:single-guide RNA (Cas9:sgRNA) ribonucleoprotein (RNP) for genome editing. The structure-activity relationship between the lipidoids and intracellular protein delivery efficiencies was explored and it was demonstrated that the newly developed LNPs are effective for gene recombination in vivo.
