Strategy for applying CRISPR/Cas9 gene editing technology in neuroscience

The gene editing technology enables cutting or/and inserting the target gene precisely. The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) system relies on two major elements, a guide RNA (gRNA) that recognizes a specific DNA sequence, and a nuclease Cas9 that cuts the target DNA and creates double-strand breaks (DSBs). Among a few gene-editing technologies available, CRISPR/Cas9 system has showed tremendous advantages over the others, mainly due to high efficiency of Cas9 and simple design of gRNA. As a versatile and powerful tool for genome engineering, the CRISPR/Cas9 technology has been used to generate genetically modified mice with unprecedented simplicity and speed, simply by timed delivery of Cas9/gRNA ribonucleoproteins (RNPs) into pronuclear-stage zygotes. The modified protocol introduces Cas9 protein instead of Cas9 mRNA by rapid electroporation, which enables the gene editing occurring before the first replication of the mouse genome, thus generating non-mosaic mutant embryos. Furthermore, it could be used to produce multiple gene mutations in a single mouse by co-delivering several gRNAs targeted to different genes. Herein, we summarize current applications of CRISPR/Cas9 technology in the field of neuroscience, and aim to provide concise information and perspectives for better utilizing this technology. In brain research, the CRISPR/Cas9 system can be applied either at the one-cell stage of the fertilized eggs in the form of Cas9/gRNA ribonucleoproteins, or at the stage of embryos or adults in the form of plasmid DNA or the viral vectors (commonly AAV variants) respectively. To deliver the system specifically into the developmental or adult brain, in utero electroporation, or stereotaxic injection is commonly employed. Several reports show that AAV-assisted Cas9/gRNA system could achieve a satisfied efficiency for genome editing in the adult mouse brain, but results vary depending on Cas9 activity, gRNA design, detection methods, and the condition of endogenous DNA repair mechanisms. Remarkable efforts have also been made to enhance the incidence of homology-directed repair for precise gene modifications. Regarding therapeutic genome editing, a couple of recent in vivo studies demonstrate that CRISPR/Cas9 system could contract or remove disease-causing alleles in animal models of certain hereditary diseases such as retinitis pigmentosa and Huntington's disease, raising hope for translating therapeutic genome editing to clinical patients. In addition, we discuss major challenges and critical improvements for this technology, including a few modifications for promoting precise editing in non-dividing cells in the case of adult brain. In the future, application of CRISPR/Cas9 technology would be enhanced greatly in neuroscience by developing cell type-specific, timed delivery system in vivo and by combining with other powerful techniques for dissecting brain neural circuits in health and disease. © 2017, Science Press. All right reserved.