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.
Highlights
Recent progress in gene-editing technologies, including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), engineered meganucleases, and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) nucleases, has greatly expanded the opportunities for accurate disruption or modification of the target genomic locus [1]
This study showed for the first time that extracellular vesicles (EVs) with dual surface nanobodies can target tumor cells and induce anti-tumor immunity by recruiting cytotoxic T cells to cancer cells
While the methods described above mostly used exosomes as EVs, Wang et al utilized a different type of EVs called arrestin domain containing protein 1- (ARRDC1) mediated microvesicles (ARMMs) [204]
Summary
Recent progress in gene-editing technologies, including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), engineered meganucleases, and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) nucleases, has greatly expanded the opportunities for accurate disruption or modification of the target genomic locus [1]. Current strategies have numerous limitations, including: (1) high molecular mass and positive charge of Cas proteins that make them difficult to package using common drug delivery tools [15]; (2) the lack of robust tissue-specific delivery vehicles suitable for cell-specific gene editing applications [16]; (3) immunogenicity [17,18,19,20,21] and other safety issues (molecular, cellular and tissue toxicity) [22] to which the majority of novel synthetic delivery vehicles are prone; and, (4) the lack of a universal CRISPR/Cas delivery platform that can be utilized for a wide array of CRISPR/Cas systems. We discuss the benefits of using EVs for gene-editing applications, and review available technologies for engineering EVs and the recent progress in CRISPR/Cas RNPs delivery using synthetic nanoparticles and EVs, synthetic or naturally produced
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