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

Abstract

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.

ENHANCED EFFICIENCY OF RNA-GUIDED CAS12a VERSUS CAS9 TRANSGENE KNOCK-IN AND ACTIVITY AT ASCHISTOSOMA MANSONIGENOME SAFE HARBOR

“…A limitation of this approach was that not every cell of egg appeared positive for the RNPs. To optimize this, alternative transfection methods such as lipid nanoparticles (97) and other methods (82,(98)(99)(100)(101)(102) could be tested. Alternatively, one could envisage to use larval stages such as sporocysts as editing templates to enhance cell-transfection efficiency (61).…”

Biomaterials-mediated CRISPR/Cas9 delivery: recent challenges and opportunities in gene therapy

“…In the context of research on infectious diseases, exosome-based CRISPR/Cas9 delivery has been suggested as a potential strategy for producing innovative therapies for viral infections including HIV and hepatitis B. For instance, a 2018 study discovered that exosomes containing CRISPR/Cas9 may target and harm HIV proviral DNA in vitro , decreasing the virus’ ability to produce genetic material ( Dubey et al, 2022 ; Mazurov et al, 2023 ). Like this, exosome-based CRISPR/Cas9 delivery was shown to effectively target the hepatitis B virus (HBV) genome in vitro and in vivo , leading to a reduction in viral replication and gene expression ( Martinez et al, 2022 ).…”