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. Display omitted