Packaging and Uncoating of CRISPR/Cas Ribonucleoproteins for Efficient Gene Editing with Viral and Non-Viral Extracellular Nanoparticles

Rapid progress in gene editing based on clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) has revolutionized functional genomic studies and genetic disease correction. While numerous gene editing applications have been easily adapted by experimental science, the clinical utility of CRISPR/Cas remains very limited due to difficulty in delivery to primary cells and possible off-target effects. The use of CRISPR in the form of a ribonucleoprotein (RNP) complex substantially reduces the time of DNA exposure to the effector nuclease and minimizes its off-target activity. The traditional electroporation and lipofection methods lack the cell-type specificity of RNP delivery, can be toxic for cells, and are less efficient when compared to nanoparticle transporters. This review focuses on CRISPR/Cas RNP packaging and delivery using retro/lentiviral particles and exosomes. First, we briefly describe the natural stages of viral and exosomal particle formation, release and entry into the target cells. This helps us understand the mechanisms of CRISPR/Cas RNP packaging and uncoating utilized by the current delivery systems, which we discuss afterward. Much attention is given to the exosomes released during viral particle production that can be passively loaded with RNPs as well as the mechanisms necessary for particle fusion, RNP release, and transportation inside the target cells. Collectively, together with specific packaging mechanisms, all these factors can substantially influence the editing efficiency of the system. Finally, we discuss ways to improve CRISPR/Cas RNP delivery using extracellular nanoparticles.

Automated summary

Rapid progress has been made in gene editing using CRISPR/Cas, but the clinical utility is limited due to delivery difficulties and potential off-target effects. Using CRISPR as a ribonucleoprotein (RNP) complex can reduce exposure time and off-target activity. Current delivery systems focus on packaging and delivering CRISPR/Cas RNPs using retro/lentiviral particles and exosomes. The mechanism of viral and exosomal particle formation, release, and entry helps to understand RNP packaging and uncoating. This review also discusses the passive loading of RNPs into exosomes and the mechanisms involved in particle fusion, RNP release, and transportation inside target cells, which can significantly influence the editing efficiency.