Gene Editing by Extracellular Vesicles

Abstract

CRISPR/Cas technologies have advanced dramatically in recent years. Many different systems with new properties have been characterized and a plethora of hybrid CRISPR/Cas systems able to modify the epigenome, regulate transcription, and correct mutations in DNA and RNA have been devised. However, practical application of CRISPR/Cas systems is severely limited by the lack of effective delivery tools. In this review, recent advances in developing vehicles for the delivery of CRISPR/Cas in the form of ribonucleoprotein complexes are outlined. Most importantly, we emphasize the use of extracellular vesicles (EVs) for CRISPR/Cas delivery and describe their unique properties: biocompatibility, safety, capacity for rational design, and ability to cross biological barriers. Available molecular tools that enable loading of desired protein and/or RNA cargo into the vesicles in a controllable manner and shape the surface of EVs for targeted delivery into specific tissues (e.g., using targeting ligands, peptides, or nanobodies) are discussed. Opportunities for both endogenous (intracellular production of CRISPR/Cas) and exogenous (post-production) loading of EVs are presented.

Keywords: biodistribution; exosomes; gene editing; nanoblades, stem cells, mesenchymal stem cells.; nanomedicines; nanoparticles; nanovesicles; pharmacokinetics.

Figure 1

Packaging Cas proteins into EVs. (A) Provisional technology based on WW-Ndfip1 interaction. Cas protein with a WW tag can be expressed intracellularly together with Ndfip1. Overexpressed Ndfip1 mediates ubiquitination of Cas-WW and promotes its loading into EVs. (B) Nanoblade technology. Virus-like particles are generated by Cas protein fused with HIV Gag and co-expressed with Gag-Pro-Pol protein. Resulting EVs exhibit minor carry-over of cytosolic matter and effectively enter target cells. (C) Provisional technology based on the fusion of a constitutive EV membrane protein (e.g., CD63) with a dimerization domain and the use of a hybrid Cas protein with another dimerization domain. Upon signal (light or a chemical molecule), domains dimerize and Cas protein is recruited into EVs. Post-production, the signal is removed, and the Cas protein is released into the EV lumina. This picture was created in BioRender.

Figure 2

Existing and prospective technologies for tagging sgRNAs to be packaged into EVs. (A) Constructing synthetic RNA chimeras with EV-targeting motifs may enrich these RNAs in EVs. (B) Using EV-enriched proteins coupled with RNA-binding domains. (C) Packaging based on the interaction of MCP protein with an MS2 aptamer introduced into cargo RNA. Palm signal localizes to the membrane of EVs together with dimerization domain DD2. MCP is fused to dimerization domain DD1. Cargo RNA interacts with MCP via MS2 aptamer. Upon incoming signal (light or a small chemical), DD1 and DD2 dimerize, bringing together all three components so that cargo RNA is packaged into EVs. After EVs are produced, DD1 and DD2 dissociate, releasing cargo RNA into the lumina of EVs. (D) sgRNA packaging device, a part of the NanoMEDIC platform. A long RNA is encoded intracellularly, comprising the Psi+ EV-localization signal and two ribozymes. Upon loading into EVs, the long construct is self-cleaved by ribozymes, releasing the sgRNA with no additional RNA sequences. Abbreviations are explained in the text. This picture was created in BioRender.