۱- Introduction

۲- Multi-layer NPs for CRISPR-Cas9 delivery

۳- Novel building blocks for multi-layer NPs

۴- Conclusions and perspective

References

Abstract

The clustered regularly interspaced short palindromic repeat (CRISPR) has great potential to revolutionize biomedical research and disease therapy. The specific and efficient genome editing strongly depends on high efficiency of delivery of the CRISPR payloads. However, optimization of CRISPR delivery vehicles still remains a major obstacle. Recently, various non-viral vectors have been utilized to deliver CRISPR tools. Many of these vectors have multi-layer structures assembled. In this review, we will introduce the development of CRISPR-Cas9 systems and their general therapeutic applications by summarizing current CRISPR-Cas9 based clinical trials. We will highlight the multi-layer nanoparticles (NPs) that have been developed to deliver CRISPR cargos in vitro and in vivo for various purposes, as well the potential building blocks of multi-layer NPs. We will also discuss the challenges in making the CRISPR tools into viable pharmaceutical products and provide potential solutions on efficiency and biosafety issues.

Introduction

In recent years, a series of programmable DNA nuclease-dependent genome editing techniques have been developed to enable efficient genetic manipulation in eukaryotes, particularly in mammalian cells [1-3]. These genome-editing techniques used for gene knock-out or knock-in predominately include meganucleases, zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the CRISPR-Cas system [4-7]. These technologies all employ endonucleases to induce double strand breaks (DSB) at intended sites in the genome, followed by DSB repair through different mechanisms including non-homologous end joining (NHEJ), homologous recombination (HR), homology-mediated end joining (HMEJ) and microhomology-mediated end joining (MMEJ) [8]. Among these genome editing techniques, CRISPR-Cas9 is a gene editing system derived from the bacterial adaptive immune system, which is capable of combating the invasive viral genome under the guidance of short sequence RNA. CRISPR-Cas9 system is highly versatile because of its simplicity and high efficiency, moreover, any genome sequence theoretically can be edited by this system. In principle, the whole genome can be site-specifically modified at the genetic level in cells and tissues with CRISPR-Cas9 system, which further allows researchers to study the relationship between gene mutations and biological phenotypes. CRISPR-Cas9 system has driven innovative applications from basic biology research to biotechnology and medicine [9].