Versatile modification of the CRISPR/Cas9 ribonucleoprotein system to facilitate in vivo application

The development of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems has created a tremendous wave that is sweeping the world of genome editing. The ribonucleoprotein (RNP) method has evolved to be the most advantageous form for in vivo application. Modification of the CRISPR/Cas9 RNP method to adapt delivery through a variety of carriers can either directly improve the stability and specificity of the gene-editing tool in vivo or indirectly endow the system with high gene-editing efficiency that induces few off-target mutations through different delivery methods. The exploration of in vivo applications mediated by various delivery methods lays the foundation for genome research and variety improvements, which is especially promising for better in vivo research in the field of translational biomedicine. In this review, we illustrate the modifiable structures of the Cas9 nuclease and single guide RNA (sgRNA), summarize the latest research progress and discuss the feasibility and advantages of various methods. The highlighted results will enhance our knowledge, stimulate extensive research and application of Cas9 and provide alternatives for the development of rational delivery carriers in multiple fields.