Recent Advances in CRISPR/Cas9 Delivery Strategies.

Bon Yip

doi:10.3390/biom10060839

Abstract

The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has revolutionized the field of gene editing. Continuous efforts in developing this technology have enabled efficient in vitro, ex vivo, and in vivo gene editing through a variety of delivery strategies. Viral vectors are commonly used in in vitro, ex vivo, and in vivo delivery systems, but they can cause insertional mutagenesis, have limited cloning capacity, and/or elicit immunologic responses. Physical delivery methods are largely restricted to in vitro and ex vivo systems, whereas chemical delivery methods require extensive optimization to improve their efficiency for in vivo gene editing. Achieving a safe and efficient in vivo delivery system for CRISPR/Cas9 remains the most challenging aspect of gene editing. Recently, extracellular vesicle-based systems were reported in various studies to deliver Cas9 in vitro and in vivo. In comparison with other methods, extracellular vesicles offer a safe, transient, and cost-effective yet efficient platform for delivery, indicating their potential for Cas9 delivery in clinical trials. In this review, we first discuss the pros and cons of different Cas9 delivery strategies. We then specifically review the development of extracellular vesicle-mediated gene editing and highlight the strengths and weaknesses of this technology.

Highlights

Clustered regularly interspaced short palindromic repeats (CRISPR) were first discovered as an adaptive immune system effector in prokaryotes [1]
The CRISPR/Cas9 system is composed of a target-specific single guide RNA and a Cas9 endonuclease
Because target recognition is based on RNA–DNA interactions, CRISPR/Cas9 has the advantages of the easy design of genomic targets and multiplexing over that of zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs)

Summary

Introduction

Clustered regularly interspaced short palindromic repeats (CRISPR) were first discovered as an adaptive immune system effector in prokaryotes [1]. By exploiting the right DNA repair pathway, CRISPR/Cas can accomplish gene disruption, deletions, knockins, or targeted editing. Choosing an appropriate delivery strategy for the CRISPR/Cas system is critical to achieving efficient and precise gene editing. If it is to be used in clinical settings, the safety profile must be considered to avoid or minimize insertional mutagenesis. The nonviral approach includes various physical and chemical delivery strategies Each of these methods has its own pros and cons that should be considered for each gene editing application (Table 1)

Viral Vectors

Nonviral Physical Methods

Nonviral Chemical Methods

Findings

Conclusions and Future Perspectives