Lipid-Nanoparticle-Based Delivery of CRISPR/Cas9 Genome-Editing Components

Abstract

Gene editing mediated by CRISPR/Cas9 systems is due to become a beneficial therapeutic option for treating genetic diseases and some cancers. However, there are challenges in delivering CRISPR components which necessitate sophisticated delivery systems for safe and effective genome editing. Lipid nanoparticles (LNPs) have become an attractive nonviral delivery platform for CRISPR-mediated genome editing due to their low immunogenicity and application flexibility. In this review, we provide a background of CRISPR-mediated gene therapy, as well as LNPs and their applicable characteristics for delivering CRISPR components. We then highlight the challenges of CRISPR delivery, which have driven the significant development of new, safe, and optimized LNP formulations in the past decade. Finally, we discuss considerations for using LNPs to deliver CRISPR and future perspectives on clinical translation of LNP-CRISPR gene editing.

Keywords: CRISPR/Cas9; gene therapy; genome editing; lipid nanoparticles; nanomedicine.

Figure 1

Visual representation of examples of different lipid nanoparticle (LNP) formulations for CRISPR/Cas9 delivery in plasmid, mRNA, and ribonucleoprotein (RNP) forms. The main components of LNP formulations are shown with specific examples of modifications used to enhance the delivery of different types of cargo. (A) Protamine is used to condense plasmid DNA (pDNA) to increase the encapsulation efficiency of pDNA within LNPs. (B) Ionizable cationic lipids conjugated with a ligand (top) and PEG lipids conjugated with antibodies (bottom) to mediate delivery to targeted cells expressing the corresponding receptors. (C) Biodegradable or bioreducible ionizable lipids are used to improve encapsulation and intracellular release with mRNA delivery. (D) Dendrimer ionizable lipids are used to enhance mRNA delivery. (E) Cationic bioreducible lipids are used in conjunction with a polyanionic macromolecule to increase encapsulation efficiency for RNP delivery. (F) Ionizable cationic lipids used in the absence of solvent enable efficient encapsulation and delivery of the active RNP complex.

Figure 2

Intracellular delivery of CRISPR/Cas9 components in plasmid, mRNA, and ribonucleoprotein (RNP) forms using lipid nanoparticles (LNPs). LNPs are taken up by cells via endocytosis (A–C). As the pH of the endosomes decreases, ionizable cationic lipids gain a positive charge and mediate cargo release. (A) Plasmid DNA (pDNA) enters the nucleus where it is transcribed by RNA polymerases. The transcribed mRNA returns to the cytoplasm where it is translated and complexes with gRNAs to form RNPs. The resulting RNPs, which contain a nuclear localization signal enter the nucleus to induce gene editing. (B) Cas9 mRNA must be translated in the cytoplasm and ultimately complex with gRNAs to form RNPs before entering the nucleus to induce gene editing. (C) LNP-mediated delivery of preformed RNP complexes can directly enter the nucleus to induce gene editing without any in cell production of Cas9.